CS248861B3 - Microcarriers suitable for growing animal cells and methods of producing them - Google Patents

Abstract

The solution concerns microcarriers from gelatin derivatives which are suitable for growing animal cells and a method of their production. The essence of the biological activity of the microcarriers according to the solution lies in the presence of aldimine binding groups in the polymeric gelatin matrix. The formation of the aldimine binding group is involved here both by the primary amino groups in the side chains of gelatin and by the amino groups of another physiologically harmless low-molecular substance, while these primary amino groups are covalently bound to the functional groups of selected aliphatic or aromatic dialdehydes. In addition, part of the dialdehyde used also causes three-dimensional crosslinking of the gelatin polymer chains, thereby achieving insolubility and infusibility of the microcarriers at higher temperatures. This then enables easy and safe sterilization of the microcarriers in the usual way in an autoclave, i.e. 15 minutes at 120 C. The microcarriers according to the solution have the form of microspheres with an average size of 100 to 300 µm, while their cultivation surface is well defined and is very suitable for laboratory or large-scale cultivation of even the most demanding types of cells, for example diploid and primary or embryonic animal cells. The essence of the method of manufacturing microcarriers according to the solution is a modification of the known process of dispersing an aqueous solution of gelatin in a mixture of suitable organic solvents. The modification of the process consists in the use of isothermal conditions during the said dispersion and in the use of well-defined substances to destroy free reactive aldehyde groups on the surface of the microspheres.

Description

The invention relates to microcarriers made of biologically active iron derivatives, prepared in the form of microspheres with an average size of 100 to 300 µm, which are suitable as a culture medium for growing animal cells.

In the industrial production of some biologically active substances, it is usually necessary to grow large amounts of cell substrate. This is the case in the production of human and animal vaccines, interferons, hormones, enzymes, nucleic acids and other biologically important substances.

Various cell culture techniques have been developed for this purpose.

Cell culture in suspension is currently the most effective technique for large-scale in vitro cell cultivation. However, only those cell cultures that do not require attachment to a solid culture substrate for their division can be grown in this way. These are mostly cell cultures with an altered set of chromosomes, e.g. stable mouse fibroblast cell lines, so-called L cells, Syrian hamster kidney cell lines, so-called BHK cells, and human cervical carcinoma cells, so-called HeLa cells, and others.

However, many cells need a suitable culture surface to divide, on which they attach and form a cell monolayer. These are mostly primary cells obtained from organs, tissues or embryos of various animals, e.g. primary cells from chicken or mouse embryos. There are also diploid cells that have twice the number of chromosomes, e.g. diploid cells obtained from the lungs of a human embryo, so-called LEP cells. These cell cultures are usually grown in large-scale using stationary techniques in large glass culture vessels, e.g. in Roux flasks with a capacity of 1,200 or 2,000 ml.

Some cell cultures can also be grown using the roller technique, i.e., in cylindrical containers in which a cell monolayer is formed over the entire cylindrical surface of the horizontally rotating culture container.

The technique of culturing cells in glass bottles is suitable in the laboratory, but not in industrial production. The disadvantages are the space requirements and the consumption of electricity in thermostats, the laboriousness in caring for a large number of culture bottles with cells, where there is also an increased risk of their contamination, and last but not least, the great laboriousness in washing the culture glass for its reuse. Therefore, increasing the volume and number of culture bottles cannot solve the need for increased production of animal cells.

A very progressive method that radically reduces production costs is the cultivation of cells on microcarriers. The principle of this cultivation is the growth and formation of a cell monolayer on microspheres, which are suspended in the culture medium and then float freely in it with stirring. The diameter of the microspheres ranges from 70 to 300 μm, optimally around 200 μm.

The advantage of this method of cell cultivation is the large cultivation area of the total surface of the microspheres on which the cells multiply. This allows obtaining a large number of cells in a relatively small volume of culture medium. With the same consumption of culture medium, the surface area available for cell anchoring increases approximately sixfold.

For example, 1 gram of microcarriers prepared based on the diethylamine derivative of dextran gel, which are already commonly produced and available on world markets, when suspended in 1 liter of culture medium, has the same surface area as 327 Petri dishes with a diameter of 10 tin, or as 21 roller culture vessels with a capacity of 1 liter.

The preparation of one production batch of viral vaccine, using the old method of stationary cultivation in bottles, requires the use of 500 Roux bottles of 1.2 liters. Each of them contains 0.12 liters of culture medium, i.e. a total of 60 liters. With the new method of cultivation on microcarriers, the same cell growth can be achieved using 250 ml of a settled suspension of microspheres with a diameter of about 200 μm, which are then dispersed in 20 liters of culture medium.

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One or two cultivation tanks, with a capacity of 20 or 10 liters, which can be easily operated by one worker, are sufficient.

Cell cultures are grown on microcarriers in culture media consisting of inorganic salts, amino acids, vitamins and coenzymes, such as in basal or minimal Eagle medium and others. The medium is usually supplemented with animal sera, such as inactivated calf serum, fetal bovine serum, or chicken serum, at a concentration of 1 to 20 i vol. In some cases, the medium is supplemented with protein hydrolysate, such as lactate albumin hydrolysate, soy peptone, yeast hydrolysate and other nutrients.

Culture media have an optimal pH of 7.0 to 7.6 and an osmolality of 260 to 350 mOsm. The temperature for culturing animal cells ranges from 35 to 40 °C, depending on the type of cell culture, in some cases an atmosphere containing 95% air and 5% carbon dioxide is used.

Microcarriers for growing animal cells are currently produced mainly from amino derivatives of agarose, dextran, cellulose or polystyrene, as well as from gelatin cross-linked with glutaraldehyde or covalently bound to the surface of agarose microspheres.

In the basic Czechoslovak patent application AO No. 247 431, various polymer compositions are described, containing a defined amount of aldimine binding groups, which significantly stimulate adhesion, growth and differentiation of animal cells.

In the case of biologically active polymer compositions in the form of microspheres according to the present invention, we start from aldimine derivatives of gelatin. The formation of aldimine binding groups involves both the free amino groups of the lysyl side groups in the polymer chains of gelatin and the primary amino groups of another physiologically harmless substance that is low molecular weight and contains at least one primary amino group in its molecule.

Examples of such substances are primarily essential or non-essential alpha-amino acids, e.g. glycine, alanine, lysine and glutamic or aspartic acid, as well as amino sugars, e.g. glucosamine or galactosamine, or it is also possible to use some biogenic amines, e.g. ethanolamine, cysteamine, serotonin, tryptamine, dopamine, histamine and other similar amines and biological activity, or other substances, e.g. 4-aminobutyric or 6-aminocaproic acid, hydroxylamine, etc.

Aldehydic functional groups of selected aliphatic or aromatic dialdehydes, such as glyoxal, glutaraldehyde, or terephthalic or ortho-phthalic acid dialdehyde, readily react with the above-mentioned primary amino groups. The resulting aldimines, so-called Schiff bases, then have the above-mentioned biological activity, i.e. they stimulate adhesion, growth and differentiation of animal cells.

The aliphatic or aromatic dialdehydes used also cause spatial crosslinking of the polymer chains of gelatin, which forms the basic matrix of the microspheres. This makes them insoluble and infusible at higher temperatures, and thus also provides the necessary shape stability. This then allows for easy and safe sterilization of the microspheres in an autoclave under usual conditions, i.e. 15 minutes at 120 °C.

The hydrolytic stability of the aldimine binding group, which is low in an acidic environment, is excellent in the pH range of 7.0 to 7.6, thus enabling the use of the described biological activity of aldimines in tissue or cell culture techniques.

However, microcarriers prepared by the method of the present invention can be hydrolytically cleaved using proteolytic enzymes that usually cleave gelatin itself, e.g.

collagenase, trypsin, dispase, etc. Enzymatic digestion can be used in cases where the isolation of the cells themselves is required. The conditions for such enzymatic digestion are generally known.

The method of preparing microspheres according to the present invention is based on the known technique described by K. Nilsson and K. Mosbach in FEBS Lett., 145-150, 1980. The principle of this method is a/ preparation of an aqueous gelatin solution, with a dry matter content of 20% by weight, at a temperature of 50 °C, b/ dispersion of the warm gelatin solution, sub a, in a mixture of organic solvents, i.e.

parts by volume of toluene and 27 parts by volume of chloroform, using 1 to 3% by volume of a suitable surfactant, e.g. glycerol monooleate, whereby this dispersion is carried out with intensive stirring and the bath of the said organic solvents is at room temperature, c/ washing the formed gelatin microspheres in excess acetone and their subsequent drying, d/ sieving the rehydrated microspheres in an aqueous solution of glutaraldehyde, with a concentration of about 5% by volume, for 30 minutes at room temperature, e/ washing the cross-linked microspheres with 0.15 M-NaCl, f/ dividing the microspheres into fractions according to their size, where the useful fraction for cell cultivation is between 100-250 g/ sterilizing the microspheres dispersed in 0.15 M-NaCl using an autoclave, i.e. 15 minutes at 120 °C, h/ the sterile microspheres are further conditioned and washed with a tenfold volume before use of the appropriate culture medium, which contains, depending on the type of cell culture, 10 to 20% vol. of fetal calf serum.

Although gelatin microcarriers prepared in this way are very suitable and well-suited for growing animal cells, the above procedure has some drawbacks:

- during the dispersion of a warm gelatin solution into a bath with a mixture of organic solvents at room temperature, the physicochemical parameters of the surface tension change significantly, mainly caused by the increase in the bath temperature during the gradual dosing of the gelatin solution, which results in a change in the size of the formed microspheres and their unusually wide distribution, the proportion of unsuitably small or large particles is thus relatively high;

- under the above conditions, the gelatin solution rapidly solidifies into a solid gel, insufficiently mixed portions of the gelatin solution form inappropriately large spheres and the proportion of spherically asymmetric or even fibrous particles also increases;

- when cross-linking gelatin microspheres with glutaraldehyde, only one aldehyde functional group is bound to the gelatin polymer chain, and the second aldehyde function remains unreacted, although still reactive, which is why the aforementioned authors use the aforementioned conditioning of microcarriers using a nutrient medium with the addition of serum;

- if the elimination of unreacted aldehyde functions is carried out in the above-mentioned manner, i.e. using an excess of nutrient medium and added serum, there is a completely undefined binding of some of its components on the surface of the microcarriers, which also results in an insufficiently defined composition of the cultivation surface of these carriers, while it is necessary to mainly take into account the fluctuations in quality and incomplete characterization of the serum used.

All of the above-mentioned shortcomings are eliminated when using the method for preparing microcarriers according to the present invention, which differs as follows:

1. Dispersion of a warm gelatin solution is carried out essentially under isothermal conditions, i.e. the bath is heated to the same temperature as the initial gelatin solution. The gelatin solution can thus be dispersed under well-defined physicochemical conditions into regular spherical microparticles, while the entire system is maintained in dynamic equilibrium, thanks to the still liquid nature of the gelatin microspheres. The size and distribution of the microspheres are determined by a number of parameters, including the density and viscosity of the gelatin solution at a given temperature, the composition of the organic phase, i.e. the mixture of organic solvents that should be isodense with the gelatin solution, the concentration of the surfactant used, the size and geometry of the reactor and the stirrer used, the speed of rotation of the stirrer, the nature of the mixing and a number of other factors,

2. The liquid system, which is maintained in dynamic equilibrium, is quickly cooled to a temperature at which the originally liquid microspheres gel and thus their final shape, size and particle size distribution are fixed, while the proportion of the required parameters of the resulting microspheres can be achieved. This makes their production very economical. The proportion of irregular particles is significantly reduced. It is necessary to emphasize here that the success of the entire operation depends on the cooling rate, because very slow cooling of the liquid system usually leads to sticking of particles and their aggregation into difficult-to-separate clusters.

3. The crosslinking of gelatin microspheres can be controlled, in addition to the concentration of the aqueous glutaraldehyde used and its volume ratio, temperature and exposure time, also by the concentration and type of the low-molecular substance used containing primary amino groups, as previously mentioned in the description of the invention. Also, the binding of unreacted aldehyde groups of glutaraldehyde, or other dialdehyde used, is carried out in a defined manner, i.e. with a known and well-defined substance, e.g. a certain alpha-amino acid, or possibly a highly diluted aqueous solution of the starting gelatin. This results in microcarriers with a well-defined cultivation surface.

Other details of the method of preparing microspheres are essentially identical to the procedure originally proposed by the above-mentioned authors.

The resulting microspheres of aldimine derivatives of gelatin, produced by the method according to the present invention, serve as a special culture substrate - a microcarrier - for growing animal cells in vitro.

The examples given below serve to illustrate the invention and do not limit the number of possible combinations, mutual representation and use of individual substances.

Example 1 grams of high-quality photogelatin were poured with 80 ml of distilled water and left to swell at laboratory temperature for 2 hours. Then the mixture was heated in a thermostat at 56 °C with gentle stirring until the swollen gelatin particles liquefied. 2 grams of glycine of p. a. purity were added to the gelatin solution with stirring and the mixture was tempered at the specified temperature for 20 minutes. The liquid solution was slowly dosed at the given temperature into a mixture of organic solvents with a total volume of 1 liter.

The dosing time was 6-8 minutes. The organic solvent bath contained 700 ml toluene, 300 ml chloroform and the appropriate amount of surfactant - PAL, which here was glycerol mono-oleate. The dispersion process took place in a 2 liter glass reactor with a “U” shape, using a three-blade stirrer of the Faundler type.

Depending on the stirring speed, the dispersion temperature and the amount of PAL, different results were obtained, which are summarized in the following table. Solid microspheres were obtained by cooling the dispersion from the indicated temperature to 20 °C, within 5-8 minutes with unchanged stirring.

Speed Time PAL Temperature Size and rotation mixing dispersion distribution min ¹ min. ml °C particles 400 15 2 20 to 22 about 500/u, 600 '· It II II very wide 800 II II II II particle distribution 1,200 II II II II 400 30 2 42 about 500 /i, wide 600 if II II 350 to 500 yen 800 II II It 250 to 500 JU 800 II 3 II 200 to 300 I 800 30 II 50 75%! 100 to 250 /1 800 It 53 85%: ». ⁿ 800 at It 56 90%: “

The advantages of the isothermal dispersion of the gelatin solution in the solvent mixture are evident from the table. The particle size range of the microspheres between 100 and 250 µm is optimal for the cultivation of animal cells on microcarriers in vitro.

Example 2

The microspheres of the aldimine derivative of gelatin prepared according to Example 1, under the conditions given in the last row of the table, were subsequently washed with a ten-fold volume of a 1% glycine solution. This reacted all remaining free aldehyde groups of the dialdehyde used. The microspheres were then transferred to a 0.15 M-NaCl solution and sterilized in a glass ampoule by autoclaving for 15 minutes at 120 °C.

Example 3

The procedure was as in Examples 1 and 2, but instead of a 1% glycine solution, a solution of glucosamine, or ethanolamine, or 6-aminocaproic acid was used under otherwise similar conditions.

Example 4

The procedure was as in Examples 1 and 2, but an excess of an aqueous solution of the starting gelatin with a concentration of 0.5% by weight was used to destroy free aldehyde functional groups.

Example 5

Comparative experiments with microcarriers based on agarose gels with covalently bound gelatin have shown that microcarriers from modified gelatin, prepared by the method according to the present invention, are at least as good as the best microcarriers on the world market.

These microcarriers are particularly suitable for growing demanding cell types, i.e. diploid and primary or embryonic. Cultivation of animal cells adhered to our microcarriers has shown that these microcarriers significantly improve the survival, growth and differentiation of these cells. For example, differentiation of embryonic chicken muscle cells - skeletal muscle myoblasts - occurs easily and successfully in myotubes.

Example 6

In suspension culture of LEP _ig cells grown on microcarriers prepared according to the procedure of Examples 1, 2 and 4, a high titer was achieved in the production of herpes virus: 1cJ/ml for type I virus and 10® ^and % ®' ⁵ /ml for type II virus.

Example 7

In the suspension culture of LEP^ _g cells adhered to microcarriers, as shown in Example 6, excellent results were achieved in the production of fibroblast interferon, 512 IU/ml of interferon were obtained, while in the old method of stationary culture only 120 IU/ml of interferon were obtained.

Claims (5)

SUBJECT OF THE INVENTION Microcarriers suitable for growing animal cells according to AO No. 247 431, characterized in that they are prepared on the basis of aldimine derivatives of gelatin in the form of microspheres of an average size of 100 and 300 µm, wherein the formation of aldimine binding moieties besides the primary amino groups contained in the starting gelatin also other fizio-safe substances, which are low molecular weight and contain at least one primary amino group in their molecule, also participate in amino groups, and these amino groups are covalently bonded to functional groups of selected aliphatic or aromatic dialdehydes so that virtually no aldehyde groups remain free in the final product . Microcarriers according to claim 1, characterized in that the low molecular weight substance containing the primary amino group in its molecule is selected from the group of compounds containing alpha-amino acids, for example glycine, alanine, lysine, glutamic or aspartic acid, amino sugars such as glucosamine or galactosamine, other amino acids , for example 4-amino-butyric acid or 6-amino-caproic acid, biogenic amines such as ethanolamine, cysteamine, serotonin, dopamine, histamine and the like. 3. Microcarriers according to claim 1, characterized in that the aliphatic dialdehyde is either glyoxal or glutaraldehyde and the aromatic dialdehyde is either ortho-phthalic dialdehyde or terephthalic dialdehyde. 4. Process for preparing microcarriers according to claims 2 to 3, characterized in that the dispersion of the aqueous solution of gelatin in organic solvents, practically immiscible with water, is carried out at a temperature above 40 [deg.] C. whereby the dispersion maintained in the dynamic equilibrium state is cooled. that the gelatin solution contained in the microspheres becomes gel. 5. Process for preparing microcarriers according to Claims 1 to 4, characterized in that the microspheres, after crosslinking with the appropriate dialdehyde and washing the free dialdehydes in excess of washing water, are conditioned by means of a dilute aqueous solution of starting gelatin below 1% by weight. the free aldehyde functions of the partially bound dialdehydes on the surface of the microspheres are safely destroyed.