DE3209127C2 - - Google Patents

Description

The invention relates to the method according to the preamble of claim 1. This The procedure is known from DE 30 12 233 A1.

Because of advances in cell biology the cells were found to be more numerous higher organisms small amounts of substances produce that is potentially or demonstrably significant for the diagnosis or treatment of Diseases. There are numerous in the literature Examples of such substances are given; this includes numerous modifiers biological response, such as hormones, Interferons and lymphokines, as well as others Substances, such as antibodies, for example in diagnostic tests are used. Cell cultures Microbiological origins have also long been for the production of antibiotics on a technical scale used.  

Especially in cell cultures derived from higher animals however, there is always a danger bacterial or other contamination. In numerous cases are also the amounts of by Cell cultures only produced the desired substance very much small, which accumulate in the culture medium, the one complex mixture of serum proteins and others Contains substances that isolate and clean the substances of interest difficult.

The invention has for its object a method for the production of interferons generated by living cells and antibodies, especially monoclonal antibodies, specify the target substances more easily isolated from conventional methods and can be obtained in higher purity at the same time. The The process is also intended to produce these substances Allow cell cultures in a serum-free medium.  

The task is performed according to the characterizing part of the patent claim solved. The subclaims relate to preferred embodiments.

In the process according to the invention, the producing cells are located in a protective microenvironment which guarantees suitable living conditions and is delimited by a semipermeable membrane which serves to separate the metabolic products from high-molecular materials which are necessary for the viability and maintenance of the cells.

The method according to the invention comprises the following steps:

• (A) Encapsulate the cells in membranes with a selected one Upper limit of permeability,
• (B) Suspend the encapsulated cells in one aqueous culture medium,
• (C) draining the metabolism of the cells in in vitro with excretion of the desired substance and
• (D) Obtaining the desired substance from the aqueous Medium or from inside the membrane;

it is characterized in that in step (A) Uses cells that produce interferons or antibodies.

According to a preferred embodiment, in step (A) Hybrid cells used monoclonal antibodies with a molecular mass above the permeability limit of the membranes generated in step (C) can be obtained from the inside of the membrane.

In the method according to the invention in step (A) beneficial for the production of interferon human fibroblasts, Leukocytes or lymphoblastoid cells used by treatment with certain viruses or high molecular weight Nucleic acids to induce interferon secretion.  

The concept of the invention has two advantages with the fact that on the one hand a protective environment for the Cells of culture are provided and other means present to the substances of interest in a medium with less added foreign components to win. According to the invention, the targeted substances of higher molecular weight serum proteins u. Like. Are separated, which is normally to support and maintain viability and the metabolism of the producing cells required are. Alternatively, the procedure of the invention also for obtaining targeted Substances in media are used that are relative small amounts of low molecular weight nutrients or Contain cell metabolism products.

In the method according to the invention, the cells in Encapsulated for nutrients, ions and other relatively low molecular weight materials permeable are essential for normal metabolism and maintenance cell viability are required. The membranes can optionally be used by the cells excreted target substance permeable or be impermeable, with an upper limit in each case of permeability that is high enough to the passage of molecules with a certain Allow selected molecular weight, generally under is about 2 · 10⁵. The capsules are then made in a usual way suspended aqueous culture medium, the normal in vitro metabolism in the encapsulated Cells can take place. Substances with a molecular mass below the upper limit of permeability the membranes that are secreted by the cells  can penetrate the membranes and collect in Culture medium. Substances with high molecular weight, such as serum proteins that are good for health and Viability of numerous types of cells higher Animals in cell cultures are required, however typically cannot be consumed themselves advantageously enclosed in the microcapsules in which they are completed and before are protected from diffusion into the culture medium. Substances that the cells use during their metabolism consume and have a molecular mass, which is low enough to pass through the capsule membranes to be able to diffuse through, emerge from the culture medium through the membranes into the capsules.

Cellular metabolites, their molecular dimensions are small enough to pass through Allow membrane to diffuse into the medium outside of the capsules.

If the targeted substances are below the Molecular mass limit of permeability possess, they diffuse into the extracapsular Medium where they are contaminating due to the lack higher molecular weight materials, as used in conventional, there are not encapsulated cell cultures, can be obtained relatively easily.

If the target substance is one above the upper limit the permeability of the membranes Has molecular mass, it collects in the Capsules, which are then separated from the medium and be broken up to obtain the substance can.  

The concept of the invention is subject to the species of the cells enclosed within the capsule membranes essentially no limitation. The concept of the invention is suitable here especially for cell cultures of tissues of all higher Animals and humans as well as microorganisms, fused Cells, for example hybridoma cells, or genetically modified cells, for example produced by the so-called emerging DNA recombinant process can also be encapsulated without difficulty will. Expressed generally, according to the invention all cell types are used, the interferons or generate antibodies and for their maintenance a suitable culture medium exists in vitro.

The invention is described below with reference to the drawings explained in more detail; show it:

Fig. 1 is a schematic representation for explaining the inventive concept and

Fig. 2 is a diagram for explaining the experimental results obtained in Example 5.

The general underlying the invention and very broad concept is to meet the individual Cells or groups of cells around a to provide a semi-permeable membrane and thus a microenvironment for the cells together with the cell culture medium to generate that through the membrane of a external aqueous medium is separated.  

Require mammalian and human cells typically to maintain health viability the presence of serum proteins, some of which have molecular weights about 65,000 to 150,000. In conventional processes the cultivation of unencapsulated cells distribute the secreted by the cells substances of interest in the culture medium and mix with the high molecular weight as well as the low molecular weight components. Because the amounts of products typically produced by the cells are quite small, the isolation is the one of interest Substances with complex cleaning processes connected. They are also mammalian cells and people are known to be more bacterial and other contamination sensitive, which is a sterile Cultivation using various Techniques for maintaining sterility are required and often the compulsion to use antibiotics in the culture medium.

Due to the inventive concept, the above explained difficulties avoided that the cells of the culture in semipermeable membranes are encapsulated, their upper limit of permeability generally not more than about 200,000 based on the molecular weight, which means that the Membranes contain pores through which substances can pass through, their maximum molecular mass at or below the upper limit of Permeability lies while using substances a molecular weight above the upper limit permeability does not penetrate the membranes can. This way it is possible to put cells together  encapsulate with a culture medium that all to maintain viability, of Metabolism and even the mitosis required Contains components, and the cells so encapsulated then suspend in a culture medium that the low molecular weight consumed by the cells Contains substances, but the necessary ones Substances with a higher molecular weight are not needs to contain.

Cells from higher organisms typically take Serum proteins and other substances with high Molecular mass does not, however, require it Are in the immediate vicinity of conservation their normal biological functions. Salts, Amino acids and other low molecular substances, which are taken up or metabolized by the cells can freely pass through the membranes and as needed by simply exchanging the culture medium located outside the capsules be supplemented.

The excreted products of cell metabolism with a molecular weight below the upper limit the permeability of the membranes accumulate in the extracapsular medium, whereby the extraction and Isolation of the metabolic products of interest is considerably simplified since none in the medium High molecular weight materials are present. The extraction of substances of interest with a molecular weight above the upper limit permeability is also according to the invention easier because these products are in the capsules accumulate and not in the extracapsular Distribute medium.  

The application of the inventive concept to cell products with low molecular weight is illustrated schematically in FIG. 1. In Fig. 1 it is shown that a cell 10 is within a capsule membrane 12 having pores 16. Substances 18 with a high molecular weight are enclosed within the capsule membrane 12 and can circulate freely in the medium 14 within the membrane boundaries. Components 20 , which are required by the cell, as well as metabolic products 22 including the substance of interest 22 'circulate freely both in the intracapsular and in the extracapsular medium and penetrate the membrane through the pores 16 . Depending on the requirements, the extracapsular medium can be removed periodically or continuously together with all of its components by suction or the like from the capsules themselves and replaced by fresh medium. The collected medium is essentially free of substances 18 with a high molecular weight, which considerably simplifies the extraction and isolation of the desired substances. The cell 10 also remains protected at all times within the intracapsular microenvironment.

In some cases, for example for stimulation the production of a particular interested Substance through the encapsulated cells, it is necessary to use cells with components to bring together with high molecular weight, whose molecular dimensions are too large to to still be able to pass through the membrane. An example of this is the production of interferon  from human fibroblasts, leukocytes or lymphoblastoid cells caused by treatment with certain viruses or high molecular weight nucleic acids to induce interferon secretion. In such cases, the cells, if the Upper limit of the permeability of the membranes smaller is as the molecular mass of the inducing Factor, before encapsulation for interferon production be excited, or the capsule membranes must be selectively broken up after cell cultivation be taking a picture of these high molecular weight Allow materials through the cells. DE 32 09 045 A1 describes a method for selective Breaking open certain types of capsule membranes stated without cell damage.

In the method according to the invention, around the cells without simultaneous negative interference their viability semipermeable membranes generated. A suitable encapsulation process is described in more detail below.

Encapsulation of the cells

The tissue or tissues to be encapsulated Cells are suspended in an aqueous medium, which is to maintain or support the ongoing metabolic processes of the special tissue or the special cell type. Therefor suitable media are commercially available. The average diameter of the material to be encapsulated can range up to a few µm vary a few mm. The best results will be however achieved with capsules, the size of which is in the range  is from 300 to 1000 µm. Individual cells, like Fibroblasts, leukocytes and lymphoblastoid cells as well as various parts or fabric units can be encapsulated if desired. Furthermore is encapsulation of microorganisms including such microorganisms possible by DNA recombinant methods or other techniques genetically were modified.

Preserving the viability of such living Materials hangs. a. of availability the necessary nutrients, oxygen transport, the absence of toxic substances in the medium and the pH of the medium. Such living materials need to be encapsulated in a physiologically acceptable environment being held. The problem is with the Adhere to membrane production conditions under which Cells and tissues can survive healthy.

In the method according to the invention, certain are determined water-soluble substances that are mixed with living cells or Tissues are physiologically acceptable, with formation a dimensionally stable, coherent mass insoluble in water made and for the production of temporary capsules or protective layers around individual cells or groups pulled around by cells; on this temporary Capsules can be treated appropriately more durable semipermeable membrane without damage of the cells are applied. Such substances are typically in a culture medium Concentration on the order of a few % By mass added, which further the cells of the culture, if necessary, serum components and optional  also cell substrates, such as collagen or other high molecular weight, materials dispersible in water, contains which serve as anchoring substrates. At Use of collagen should increase concentration in the range of about 10 µg / ml to about 1 mg / ml and preferably in the order of magnitude 100 up to 500 µg / ml.

The solution is then converted into droplets, which containing the cells along with the medium and then immediately made water insoluble and in Gelform be converted, at least in one Surface layer. Then the shape-retaining temporary capsules with a more permanent one Membrane provided, which then if necessary with the release of the capsule contents be selectively broken up without damage can. After creating the permanent membrane, you can the capsule contents, if that's to create the temporary Capsules used material allow this again be liquefied. This is done through restoration the conditions in the medium under which the Material is soluble.

The one used to create the temporary capsules Material can be any non-toxic, be water soluble material by change the ionic environment or the ion concentration be converted into a dimensionally stable mass can. The material should also be numerous light ionizable anionic groups, for example Carboxyl groups, by training  of salt bonds can react with polymers, which have a large number of cationic groups. As explained in more detail below, such Materials without difficulty the deposition a permanent membrane of selectable permeability on the surface of the temporary capsules.

Among the preferred materials for creating the Temporary capsules include acidic, water-soluble natural ones or synthetic polysaccharide gum. Such Materials are commercially available. you will be typically extracted from plant materials and used in many cases as food additives. As a preferred water-soluble rubber Sodium alginate used. Alginates in the molecular mass range of 150,000 can be used; due to however, their molecular dimensions can usually be the final capsule membranes do not penetrate. Therefore, alginates are lower Molecular mass, for example with molecular masses from 40,000 to 80,000, easier through diffusion through a membrane of suitable porosity from the remove intracapsular volume and therefore preferred. Other gums that can be used are acidic Fractions of guar gum, carageenan, pectin, Tragacanth and xanthan gum.

These materials contain glycosidically cross-linked Saccharide chains. Your free acid groups are often in the form of alkali metal salts, for example in the sodium form. When a polyvalent ion, such as calcium or aluminum, against the alkali metal ions are exchanged the water-soluble polysaccharide molecules below  Formation of a water-insoluble, dimensionally stable Gels cross-linked by separating the ions by ion exchange or with a release agent or be made soluble again with a masking agent can. Although essentially any multi-valued ones Ions that form salt with the acid Rubber are able to be used are preferably physiologically acceptable Ions, for example calcium, applied, whereby tissue can be kept alive. However, other polyvalent cations can also be used. Magnesium ions are on the other hand Gel formation with sodium alginate ineffective.

A typical solution is composed that they have equal volumes of a cell culture in the corresponding Medium and a 1 to 2% solution of the gum in physiological saline. When using sodium alginate, one has 1.2 to 1.6% solution proved to be cheap.

When the cells to be encapsulated bind on an anchoring substrate Cell division takes place, and the cells within of the capsules to be cultivated can collagen or another natural or synthetic, water-dispersible protein or polypeptide present in the cell culture and within the intracapsular Volume of the capsules finally produced be included. If a polymer with numerous cationic groups, for example polylysine, for this  is used, the cationic groups react with the anionic sites on the water-soluble rubber to form an essentially water-insoluble Matrix linked to the rubber. The preferred Concentrations are for such materials in the range of the order of 100 to 500 µg / ml the suspension including the rubber solution.

In the next step of the encapsulation process the rubber solution containing the cell culture is in Droplets of a desired size are converted. After that the droplets are immediately generating dimensionally stable masses that preferably but not necessarily in spherical or spheroidal form. The drop formation can be done in a conventional manner.

A tube containing an aqueous saline solution with polyvalent Cations, for example a 1.5% CaCl₂ solution, contains, is provided with a stopper that a Drop generating device carries. The drop generator has a housing with a upper air inlet nozzle and an elongated hollow body on which is provided in the plug in sliding fit is. A 10 ml syringe is attached to the top of the housing a step pump mounted, for example one with PTFE coated needle of 0.25 mm Has inner diameter that runs longitudinally through the housing goes through. The inside of the case is constructed so that a constant at the end of the needle laminar air flow acts.  

In operation, the syringe is the one to be encapsulated Material containing solution filled and the step pump actuated, the batch solution droplets the needle tip. The droplets will torn off by the air flow and fall about 2.5 cm deep into the CaCl₂ solution, where it is absorbed by Calcium ions can be gelled immediately. The distance between the needle tip and the surface of the CaCl₂ solution is large enough so that the Sodium alginate cell suspension which is physically take the cheapest shape, namely the spherical shape can, at the maximum volume at the same time minimal Surface is present. The air inside the Rohrs flows out through an opening in the stopper. This procedure crosslinks the gel, and highly viscous, dimensionally stable form temporary protective capsules that hold the suspended Contain cells and their medium. Collect the capsules itself in the solution as a separate phase separated by suction.

In the next stage of the process, a semipermeable Membrane on the surface of the temporary Capsules by cross-linking the surface layers upset. This can be done in that the gelled temporary capsules in an aqueous solution of a polymer containing cationic groups be that with the anionic functional Groups in the gel molecules can react. Polymers, the groups reactive towards acid groups, such as such as free imino or amino groups, are preferred. At this stage the polysaccharide rubber through interaction between the  Carboxyl groups and the amino or imino groups over Networked salt bonds. The permeability can be advantageous within certain limits by selection the molecular weight of the crosslinking polymer used and by adjusting the concentration of the polymer solution and the duration of exposure. Low molecular weight polymer solutions penetrate further for a given period of time the temporary capsules as solutions of polymers with higher molecular mass. The degree of penetration of the Crosslinking agent can be with the resulting Correlate permeability. In general, the The higher the molecular weight, the larger the pore size and the lower the penetration is. General can polymers with a molecular weight in the range of 3000 to 100,000 or more depending on the reaction time, the concentration of the polymer solution and the desired permeability can be used.

Favorable reaction conditions are e.g. B. before if polylysine with an average molecular weight of used about 35,000, the reaction below Stirring is carried out for 2 min and a physiological Saline solution is used, the 0.0167% Contains polylysine. This allows membranes with an upper limit of permeability of about 100,000 be achieved. Optimal reaction conditions that yourself to adjust the permeability in a given  System may be due to the above given guidelines easily determined empirically will. With this procedure it is possible to Upper limit of the permeability of the membranes set a selected value below about 200,000.

Examples of suitable crosslinking polymers proteins and polypeptides are natural or synthetic origin with free amino or imino groups, polyethylene amines, polyethylene imines and polyvinylamines. Polylysine is suitable both in the D and L form. Other polyamino acids, such as polyarginine, polycitrulline and polyornithine can also be used. Polymers with higher positive charge density, for example, polyvinylamines adhere strongly the anionic groups of the gel molecules and form stable membranes, which, however, are relatively difficult are to be broken open.

By treatment with a dilute rubber solution the free amino groups on the surfaces of the capsules, which otherwise tend to clump together of capsules.

At this stage you can encapsulate Capsules are obtained that are semipermeable Membrane that contain a gelled rubber solution, a compatible with the corresponding cell type Culture medium and the cells and, if necessary, an internal  Matrix made from collagen or other anchoring substrate includes. Since the mass transport within the Capsules and promoted through the membranes it should be preferred to close the gel again to liquefy its water-soluble form. This can be done by restoring the conditions under which the rubber is liquid, for example by removing the calcium or other multivalent cations from the inner gel. The medium in the capsule can be easily by immersing the capsules in phosphate buffered Saline solution, the alkali metal ions and hydrogen ions contains to be resolubilized. When inserting the capsules in the solution with stirring are monovalent Ions against calcium or other multivalent Ions exchanged within the rubber. At the same Sodium citrate solutions can also be used for this purpose to separate the divalent ions or to complex.

The cell cultures encapsulated as above suspended in culture media, all with the demands associated with the respective cells specifically meet and in which the cells their continue normal in vitro metabolism. If the Culture an environment of high molecular components, such as serum components, these can can be omitted from the extracapsular medium. The components normally taken up by cells typically have a relatively lower one Molecular mass and diffuse easily through the Capsule membranes in the microenvironment of the cells,  where they penetrate the cell membrane. The metabolic products of cells in the intracapsular medium are deposited in, diffuse when they a molecular weight below the upper limit of Also have permeability of the capsule membranes through and collect in the extracapsular medium at.

The encapsulated cells can under conditions for example the temperature, the pH or the Ionic environment are cultivated, the same are like traditional cultures. Even that of The cells produced products can be made from the extracapsular medium or from the capsules known methods can be obtained.

The method according to the invention offers the following Advantages:

• 1. The cells of the culture are before shear forces and mechanical damage as well as contamination protected by substances whose dimensions above the upper limit of the permeability of the membranes lie. This means that the requirements in handling and sterility compared to previous ones Culture procedures are not as strict as microorganisms do not reach the encapsulated cells can and virus-infected cells not necessarily also lead to infection of the remaining cells.
• 2. The capsules in turn immobilize the Cells within an environment in which  High molecular weight materials included are, at the same time nutrients with lower Molecular mass easily introduced as well as the products can be easily removed. This can the nutrient solution is collected batchwise or continuously and supplemented if necessary, without disturbing the cells.
• 3. The targeted ones produced by the cells Substances can be easily obtained. The secluded Cell products with molecular dimensions, that are small enough to encapsulate the capsule membranes to be able to penetrate collect together with the nutrients in the extracapsular medium. High molecular weight components however, are not in the extracapsular medium submitted, thereby attracting the interested Cell products is significantly simplified. Separated cell products, their molecular dimensions above the upper limit of permeability of the membranes lie within of the capsules. Cell products not by the Cell membrane can be released through of course also be of interest. Such products can be relatively concentrated Form by isolating the capsules and then selective breaking of the capsule membranes, for example by the procedure described below, and possibly by breaking open the cell membranes be won.  
• 4. The intracapsular volume represents an environment which is well suited for cell division. For suspension cultures it was found that within the capsules mitosis occurs. Anchoring dependent Cells in normal cultures in one grow, multiply two-dimensional monolayer forming aggregates within the capsules. Such cells use the inner surfaces of the membranes as a substrate and / or settle on the high-molecular materials explained above firmly inside the capsules. This leads to a significant increase in cell density compared to traditional cultures. The conservation the viability of such cell clusters is supported by the fact that the ratio of The surface area of the capsules is very large is so that all cells have access to the necessary nutrients, oxygen and. the like.

In certain cases it can be advantageous the capsule membranes to release the cells without Selectively destroy damage. One in this regard manufacturing is an important example by interferon. Capable of interferon production Cells need to achieve the level of interferon production exposure to certain viruses or Nucleic acids are exposed. With some procedures to induce interferon production culture inhibitors for inhibition added to protein synthesis. The growth stage  The cultivation process must therefore be carried out under conditions be carried out which is significant on the conditions of induction of the Differentiate interferon production. If the for Induction of interferon production applied Substances have a molecular mass above the upper limit of the permeability of the capsule membranes lies like this through induction Viruses can induce interferon production not in the encapsulated cell culture be made. Interferon producing Cells must therefore, if within Capsules were cultivated by breaking open the Membranes are released to interferon production to be able to induce.

Breaking open the membranes

Enclosed in membranes of the type indicated above Cells can be released by a procedure with the commercially available reagents with properties that are applied encapsulated cells not significantly adversely affect. The capsules are first separated from the suspending medium for removal present on the outside of the microcapsules contaminating materials thoroughly washed and then with stirring in a Solution with monatomic, multivalent cations, such as Calcium ions, and a "stripping" polymer  with numerous anionic groups, such as such as a salt of a polysulfonic acid or polyphosphoric acid, dispersed. At this stage it is Use of heparin, i. H. a natural sulfonated Polysaccharides, preferred. The anionic charge density of the separation polymer used should be equal to or preferably greater than the charge density originally for membrane production applied polyanionic material. The molecular mass of the polymer should match the molecular weight of that used in membrane production Polymers with numerous cationic groups at least comparable and preferably larger be.

Within the suspension of capsules in the Mixed solution, the calcium ions compete with the cationic Groups of the polycationic used for membrane production Polymer chains around the anionic groups on the water soluble rubber. Compete at the same time the heparin or other polymers with numerous anionic groups dissolved in the solution with the rubber in the membrane around the cationic Groups of polymer chains. This leads to an in Water dispersible or preferably water soluble Complex, for example made of polylysine and heparin, as well as for the association of monatomic Cations with the molecules of the gel.

Through this step, the Membranes when subsequently brought into contact with a Release agent or a masking agent is soluble because of this  the breaking of the membranes by absorption monatomic ions from the gel is completed. Remains of capsule membranes remaining in the medium can be easily separated from the cells if necessary.

A commonly preferred solution for the first stage of selectively breaking the membranes contains 1.1% (w / v) calcium chloride and 500 to 1500 units of heparin / ml solution. This solution is mixed with such a volume of microcapsules that they take up about 20 to 30% of the total volume of the suspension. Calcium chloride and heparin are preferred for breaking open capsules containing cells, since both reagents are physiologically compatible with most cells and this minimizes the possibility of cell damage. Mixtures of aluminum salts or other salts with polyvalent cations (but not Mg ⁺⁺ ions) can also be used together with the polysulfonic acid or polyphosphoric acid salt of the type specified above.

The concentrations of at this stage of the process used monatomic ions and the anionic polymers can be used in a wide range vary. Optimal concentrations can can be easily determined empirically and depend on the exposure time and the time needed to generate the Membranes used each special polymer from.  

As a release agent for selective breaking up of the membranes is usually preferred sodium citrate used, however, other alkali metal citrates and EDTA alkali salts are used. The optimum is when using sodium citrate Concentration on the order of 55 mM. To minimize of cell damage, it is preferred that Citrate or other release agents or masking agents use dissolved in isotonic saline.

The invention is described below using exemplary embodiments explained in more detail.

example 1 Production of interferon- β

Through 5 min treatment of human foreskin tissue with trypsin and EDTA at 37 ° C in known Fibroblasts obtained in this manner were analyzed in a complete culture medium (CMLR 1969) suspended, calf fetal serum purified with 40 vol.%, 0.8% sodium alginate and purified calf skin collagen added in a concentration of 200 µg / ml was. The density of the cell suspension was approximately 1.5 x 10⁷ cells / ml.

The temporary alginate capsules were as follows generated.

A 1.5% calcium chloride solution was used around the droplets of the cell suspension like explained above to gel. The droplets with a diameter of the order of 300 to 400 microns coming from the tip of the needle of the drop generator emerged, gelled instantly  in the calcium chloride solution. The gelled Capsules were then transferred to a beaker, the Contained 15 ml of a solution consisting of 1 part of a 2% buffer solution of 2-cyclohexylaminoethanesulfonic acid in 0.6% NaCl solution (isotonic, pH = 8.2) and 20 parts of 1% CaCl₂ solution. After 3 minutes of immersion, the capsules were put in twice 1% CaCl₂ solution washed.

On the surface layers of the capsules were then by 3 min suspension in a 0.005% (M / V) solution of polylysine (molecular mass 43,000) semipermeable Membranes deposited.

The resulting capsules were in the culture medium, which was supplemented with 10% calf fetal serum, suspended. The above steps were carried out at 37 ° C. After incubation at the same temperature microscopic examination revealed that the capsules contained fibroblasts in which cell division had taken place and that within the Microcapsules the three-dimensional fibroblast morphology exhibited.

4 to 5 d after the incubation, the encapsulated fibroblasts were induced by the Vilcek method to produce interferon- β (INF- β ) by the superinduction method. The capsule suspension was in an atmosphere with 5% CO₂ (95% air) for 1 h at 37 ° C in the presence of 100 µg / ml poly I-poly C (double-stranded RNA) as an INF- β inducer and 50 µg / ml cycloheximide suspended as an inhibitor of protein synthesis. After 1 h, the suspended capsules were washed with culture medium containing 50 μg / ml cycloheximide, and then suspended in the same solution at 37 ° C. under a 5% CO₂ atmosphere for 3 h. After the incubation period, the washing step was repeated, whereupon the capsules were resuspended in culture medium containing 50 μm / ml cycloheximide and 5 μg / ml actinomycin D as an inhibitor of RNA synthesis and 2 h at 37 ° C. under 5% CO₂ Atmosphere were incubated.

The capsules were then washed twice with the culture medium and then suspended in serum-free medium at 37 ° C. for 18 to 24 h, during which time the fibroblasts excreted INF- β , which had a molecular mass of the order of 21,000, and from the extracapsular medium could be won.

Example 2 Production of interferon- β

The procedure was as in Example 1 with the Difference that the capsule membranes before induction were selectively broken up so that the Poly I-Poly C easier to get to the fibroblasts could. The breaking up of the capsules was as follows carried out.

10 ml portions of the microcapsule suspensions, containing 500 to 1000 capsules / ml were discarded left whereupon the suspension medium was sucked off. The capsules were then taken twice washed with phosphate buffered saline (pH = 7.4). The washed capsules were then with a 3.0 ml aliquot of the phosphate buffered Saline mixed, the 1000 units  Heparin / ml and 1.1% (M / V) CaCl₂ contained. The Suspension was stirred for 3 min at 37 ° C; subsequently the capsules were allowed to sit down and after aspirating the supernatant twice with 3.0 ml of 0.15 M NaCl solution washed. After suction The second wash solution became the capsules with 2.0 ml of a solution from equal volumes 110 mM Sodium citrate solution and 0.15 M NaCl solution (pH = 7.4) mixed. The mixture was swirled by hand for 1 min. in order to dissolve the membranes bring. The cells were then cut twice washed with the medium.

The cells were then suspended in the medium and subjected to the process for inducing INF- β production given in Example 1 and finally encapsulated as in Example 1. The capsule suspension obtained was then incubated in a serum-free medium for 18 to 24 hours, during which time the cells excreted INF- β , which diffused through the capsule membrane and accumulated in the extracapsular medium.

When using poly I-poly C with a sedimentation number of 5 S (poly I and poly C connected in the form of a double-stranded RNA), the following production amounts of INF- β , expressed as units INF- β / 10⁵ cells, were in Examples 1 and 2 , achieved:

When using Poly I - Poly C with a sedimentation number of 12 S (double-stranded, as in the trade), according to Examples 1 and 2, the same production quantities of INF- β as stated above were achieved.

The 100-fold increase in production volume in the procedure of Example 2 Example 1 is probably based at least in part that the poly I - poly C in the example 2 better applied to the Cells could get.

Example 3 Production of interferon- β

The procedure was as in Example 1, with the difference that capsules which did not contain collagen were used. The encapsulated cells were cultivated in the usual monolayer culture, treated with trypsin and induced with poly I - poly C (sedimentation number 5 S) and at the same time microencapsulated. In this experiment, it was found that the extracapsular medium contained 2500 units of INF- β / 10⁵ cells.

Example 4 Production of monoclonal antibodies

Hybridoma cells produced by fusion of Mouse spleen cells and mouse myeloma cells in known Were made to a cell density of 3.0 × 10⁶ cells / ml. The hybridoma cells represented a cell line capable of reproduction, in whose  Cultivation of antibodies against dinitrophenyl bovine serum albumin were generated. 3 ml aliquots of Cell suspension was prepared by adding 2.1 ml of a suspension, containing 1.4% sodium alginate at 0.6 ml Calf fetal serum and 0.3 ml physiological saline (150 mM). Droplet of this Suspension were immediately in CaCl₂ solution gelled and then with a 0.016 mass% Solution treated by poly-L-lysine. The inside of the resulting capsules were then immersed for 6 min in a solution of 1 part 110 mM sodium citrate solution and 3 parts of 150 mM saline again liquefied. The hybrid cells containing Capsules were then mixed suspended from RPMI-1640 medium, the 20% calf fetal serum inactivated by heating contained.

Cell counts of the encapsulated and non-encapsulated hybridoma cell cultures and the determination of the amount of monoclonal antibodies which were generated by the encapsulated and the non-encapsulated culture were carried out periodically. The results obtained are shown graphically in FIG. 2.

Example 5 Production of interferon- α from leukocytes

30 ml of white blood cells were mixed with 3.0 ml 5% EDTA solution treated and repeated 10 min treated with 0.83% NH₄Cl solution at 4 ° C to  to lyse the blood cells. By centrifugation for 5 min at 4 ° C between treatments with NH₄Cl solution were the fragments of the remaining ones intact leukocytes separated. The cells were initially in MEM medium (serum-free medium) diluted 100-fold and 15 min with tryptan blue colored. One performed on a sample Cell count showed that about 1.3 · 10⁹ leukocytes / 30 ml of the material used had survived.

The cells then became cell density of 1 × 10⁷ cells / ml suspended in the medium, that with 2% inactivated by heating Calf fetal serum was supplemented.

The induction of interferon production was carried out by contacting the cell suspension with Sendai virus in various concentrations (in hemagglutination units / ml) for 1 h at 37 ° C. with stirring. The viruses were then centrifuged at room temperature from the cells, which in turn were resuspended in equal volumes of MEM medium with 4% calf fetal serum inactivated by heating and 1.4% sodium alginate solution. Then capsules were produced according to the method explained above, which were resuspended in serum-free and in serum-containing medium. There were no significant differences in the amounts of interferon- α (INF- α ) that were detected in the extracapsular medium of these test samples. The production quantities of INF- α were the same as for the non-encapsulated reference cultures. The following results were obtained in these experiments:

Units of Sendai virus Generated amount of INF- α (HA units / ml) (INF- α units / 10⁷ cells)

60010 30020 15033 7550

Example 6 Production of interferon- α from lymphoblastoid cells

Namalwa cells were cultured in conventional culture as well as in microcapsules in RPMI 1640 medium containing 10% calf fetal serum inactivated by heating. These cell suspensions were then subjected to the induction of INF- α production and of INF- α production itself, one cell suspension being encapsulated and the other not being encapsulated. The cultures contained approximately the same number of cells. The encapsulated and the non-encapsulated culture were mixed with bromodeoxyuridine in a concentration of 25 mg / ml in double-distilled water to inhibit mitosis. After 36 h incubation at 37 ° C, the cells of both cultures were washed and resuspended in culture medium supplemented with 2% calf fetal serum inactivated by heating.

The encapsulated culture then underwent treatment for the selective opening of the capsule membranes subjected. The capsules were taken three times washed in a physiological saline solution 1.1% CaCl₂ containing heparin solution Incubate 1000 units of heparin / ml for 10 min at 37 ° C and then washed again with brine. The washed capsules were then at 5 min 37 ° C with sodium citrate in physiological saline incubated. By stirring the capsule suspension at this stage the membranes detached and release the cells. The Cell suspension was then removed centrifuged from fragments and several times with Washed citrate saline.

The two cultures were then in fresh culture medium suspended with 2% calf fetal serum inactivated by heating and buffer (pH = 7.4) was added, the cell density Was 1.0 x 10⁶ cells / ml.

The traditional culture as well as the encapsulated one Culture was then carried out with Newcastle disease virus (Bankowski strain) in amniotic fluid.

The virus concentration was 1.0 · 10⁸ pfu / ml. 1 ml of the virus suspension was made up to 10 ml each added to the cell suspension. The cultures were  then incubated at 37 ° C for 24 h.

The conventional culture then became divided into 5 parts (see the following Points 1. to 5.); became the encapsulated culture divided into 4 parts (see the following Points 6. to 9.). Eight of the nine aliquots of cultures were treated as follows:

Sample 1: Untreated. Sample 2: In culture medium at 2% by heating inactivated calf fetal serum resuspended. Sample 3: Resuspended in serum-free culture medium. Sample 4: With 5% culture medium by heating encapsulated inactivated calf fetal serum; Capsules suspended in serum-free medium. Sample 5: With 5% culture medium by heating encapsulated inactivated calf fetal serum; Capsules in medium with 2% calf fetal serum suspended. Sample 6: Resuspended in serum-free culture medium. Sample 7: In culture medium with 2% inactivated by heating Calf fetal serum resuspended. Sample 8: With 5% culture medium inactivated by heating Calf fetal serum again encapsulated; Capsules in serum-free culture medium suspended.  Sample 9: With culture medium with heating inactivated Calf fetal serum again encapsulated; Capsules in culture medium with 2% calf fetal serum suspended.

The nine cultures were then examined for their INF- α production.

The table below shows the number of cells required to produce 1 unit INF- α in the cell cultures 1 to 9 concerned.

Sample No. Required cell number

130 245 3- 4680 52000 640 740 81000, 360 9200, 100.

Claims (4)

1. Process for the production of substances produced by living cells

• (A) encapsulating the cells in membranes with a selected upper limit of permeability,
• (B) suspending the encapsulated cells in an aqueous culture medium,
• (C) allowing the metabolism of the cells to run in vitro with the excretion of the desired substance and
• (D) obtaining the desired substance from the aqueous medium or from the inside of the membrane,

characterized in that cells are used in step (A) which produce interferons or antibodies. 2. The method according to claim 1, characterized in that when using hybridoma cells monoclonal antibodies with a molecular mass above the permeability limit of the membranes and these from the inside of the membrane be won out. 3. The method according to claim 1, characterized in that he  for the production of interferon human fibroblasts, Leukocytes or lymphoblastoid cells caused by treatment with certain viruses or high molecular weight Nucleic acids are induced to interferon secretion, starts.