CH654328A5 - METHOD FOR PRODUCING SUBSTANCES GENERATED BY LIVING CELLS. - Google Patents

Description

The present invention relates to a method for producing substances produced by living cells.

Advances in cell biology have shown that the cells of various higher organisms produce small amounts of substances that have significant potential or demonstrable utility for the treatment or diagnosis of diseases. Examples of such substances are abundant in the literature and include various biological response modifiers such as hormones, interferons and lymphokines, as well as other substances such as antibodies used in diagnostic tests. Cell cultures of microbial origin have long been used to produce large quantities of antibiotics.

Especially with cell cultures that come from higher animals, there is a constant risk of bacterial or other contamination. In most cases, the amounts of the substance of interest generated by the cell cultures are also very small and accumulate in the culture medium, which contains a complex mixture of serum proteins and other substances. This makes isolation and purification of the substance of interest difficult.

The invention provides a method for producing substances that are produced by living cells. Practical implementation of the invention has the double advantage that it provides a protective environment for the cells of the culture and a means for collecting substances of interest in a medium which contains fewer admixed foreign components. The invention can be used to separate the substance of interest from the higher molecular weight serum proteins and the like that are normally required5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

3rd

654 328

are loaned to maintain the continued viability and metabolism of the producing cells. However, the invention can also be used to collect the substance of interest in a medium which contains relatively small amounts of low molecular weight nutrients or cell metabolism products.

The method involves encapsulating cells within a membrane that is permeable to the nutrients, ions, and other relatively low molecular weight materials required to maintain normal metabolism and continued cell viability. The membrane may or may not be permeable to the substance of interest excreted by the cells, but in any case has an upper limit of permeability sufficient to allow the passage of molecules with a molecular weight of a certain predetermined value, generally below about 2.0 x IO5 u lies. The capsules produced in this way are suspended in a conventional aqueous culture medium and the encapsulated cells are allowed to experience the normal in vitro metabolism. Substances with a molecular weight below the upper permeability limit of the membrane, which are secreted by the cells, penetrate the membrane and accumulate in the culture medium. Advantageously, high molecular weight substances, such as serum proteins, which are necessary for the health and viability of many types of cell cultures from higher animals, but which are typically not consumed themselves, can be incorporated into the microcapsules, where they are enclosed and do not diffuse into the culture medium can. Substances that consume cell culture during metabolism and that have a molecular weight that is low enough to allow diffusion through the capsule membranes pass from the culture medium through the membranes. Metabolic products of the cells with sufficiently small molecular dimensions that they can pass through the membranes diffuse into the medium outside the capsule. If the substances of interest have a molecular weight below v the upper limit of permeability, they diffuse into the medium outside the capsules, from which they are harvested relatively easily because of the absence of contaminating higher molecular weight materials which are present in non-encapsulated cell cultures according to the prior art can be. If the substance of interest has a molecular weight above the upper limit of the permeability of the membranes, then it accumulates in the capsules, which can then be isolated from the medium and destroyed so that recovery processes can be used.

The invention is essentially not subject to any restrictions on the types of cells that can be enclosed within the capsule membranes. Specifically, it is contemplated that cultures from the cells of tissues of all higher animals as well as microorganisms can be used. Fused cells, e.g. Hybrid dom cells, or genetically modified cells, e.g. using the evolving technology of recombinant DNA can also be encapsulated without difficulty. In short, if a culture medium exists which is useful for obtaining the cell type in question in vitro, this cell type can be used according to the invention. Examples of the types of substances which can be produced by means of the method according to the invention are insulin, glycogen, prolactin, somatostatin, thyroxine, steroid hormones, pituitary hormones, interferons, follicle-stimulating hormones (FSH), PTH and antibodies.

The product of step (B) comprises encapsulated viable cells suspended in an aqueous culture medium. The encapsulated cells have membranes characterized by an upper limit of permeability sufficient to allow the passage of the nutrients necessary for cell metabolism and continued viability. The membranes enclose viable cells which are arranged in a medium which contains all components which are necessary to maintain the metabolism of the cells and which are of a size in the range above the upper permeability limit of the membrane. The culture medium contains components that are necessary to maintain cell viability and that have a molecular weight below the upper permeability limit of the membranes 15.

The aim of the invention is therefore to provide a process for the production of substances produced by living cells using a system in which the producing cells are contained in a protective, healthy microenvironment 20 which is delimited by a semipermeable membrane which serves to produce products to separate the cell metabolism from high-molecular materials, which are necessary for the viability and maintenance of the cells, an improved method for the production of biologically active materials from 25 cell cultures as well as the production of antibodies and agents modifying biological reactions, such as hormones, interferons and lymphokines, in a serum-free medium.

The invention is described in the following description and 30 of the drawing, in which FIG. 1 is a schematic diagram which explains the inventive concept, and FIG. 2 is a graphical representation which shows the results of the experiment described in Example 5.

The general idea of the invention is to provide a semipermeable membrane around individual cells or groups of cells in order to provide a micro-milieu for the cells, which is complete with the cell culture medium and is separated from an external aqueous medium by the membrane. Human or mammalian 40 cells typically require the presence of serum proteins, some of which have a molecular weight in excess of 65,000 to 150,000 µ, for continued health and viability. In the prior art method of growing non-encapsulated cells, the materials of interest that are secreted by the cells are dispersed in the culture medium and mixed with both high molecular weight and low molecular weight components. Since the amounts of products produced by the cells are typically quite small, isolating the substance of interest becomes a difficult cleaning task. Furthermore, human and mammalian cell cultures are known to be sensitive to contamination from bacterial or other sources. This requires that the breeding be carried out using various methods of maintaining sterility and that antibiotics often have to be included in the medium.

By practicing the invention, the above difficulties are alleviated by encapsulating the 60 cells of the culture within semipermeable membranes with a predetermined permeability limit which is generally no higher than about 200,000 µ, that is, the membrane contains pores that it Substances with a maximum molecular weight at or below the upper 65 permeability limit enable the membrane to be passed through, while substances with a molecular weight above the upper permeability limit cannot cross the membrane. This enables the encapsulation of

654 328

4th

Cells together with a culture medium that contains all the components necessary for ongoing viability, metabolism and even for mitosis, and then to encapsulate the cells encapsulated in a culture medium that contains low-molecular substances that are consumed by the cells, but that does not need to contain the required high molecular weight substances.

Typically, cells from higher organisms do not take up high molecular weight serum proteins and the like, but need them in close proximity to continue their normal biological reactions. Salts, amino acids and other low-molecular factors, which are taken up by the cells or are subjected to metabolism, pass freely through the membrane and can be supplemented as required simply by changing the culture medium located outside the capsule. Eliminated products of cell metabolism with a molecular weight below the upper limit of the permeability of the membrane accumulate in the medium outside the capsules, from which the metabolic products of interest can be harvested and isolated more easily because there are no high molecular weight materials in the medium. The harvesting of products of interest with a molecular weight above the upper permeability limit is also facilitated, since such products accumulate inside the capsules and are not distributed in the large volume present outside the capsules.

The idea of the invention, applied to low molecular weight cell products, is schematically illustrated in Fig. 1 of the drawing. As shown, a cell 10 is arranged within a capsule membrane 12 with pores 16. High molecular weight factors 18 are enclosed within the membrane 12 and can circulate freely within the medium 14 within the limits of the membrane. The components 20 required by the cell and the metabolic products 22 including the substance of interest 22 'circulate freely both in the medium inside the capsules and in the medium outside the capsules and cross the membrane through the pores 16. This can be done outside the capsules if required located medium either periodically or continuously with all its components by suction or the like from the capsules themselves and replaced by fresh medium. The collected medium is essentially free of high molecular weight components 18, which simplifies the harvesting and isolation processes. Furthermore, the cell 10 remains protected at all times within the micromilieu within the capsules.

In some cases, e.g. In order to stimulate the production of a special substance of interest by encapsulated cells, it is necessary to expose the cells to high molecular weight components, the molecular dimensions of which are too large to cross the membrane. One example is the generation of interferon from human fibroblasts, leukocytes or lymphoblastoid cells, which are caused to excrete interferon by treatment with certain viruses or high-molecular nucleic acids. If the upper permeability limit of the membranes is lower than the molecular weight of the secretion-causing factor, the cells must be subjected to interferon induction before encapsulation, or the capsule membranes must be selectively destroyed after the cells have been grown in order for such high molecular weight materials to be released from the cell can be included. U.S. Patent Application No. 243,584, March 13, 1981, discloses a method for selectively destroying certain types of capsule membranes that can be used for these and other purposes without damaging the cells.

The method of the invention depends on the ability to form semipermeable membranes around cells without adversely affecting their continued viability. One of the suitable encapsulation methods is described in detail below.

The tissue or cells to be encapsulated are suspended in an aqueous medium that is suitable for maintaining or maintaining the ongoing metabolic processes of the particular tissue or cell type. Media suitable for this purpose are commercially available. The average diameter of the material to be encapsulated can vary within wide limits between a few micrometers and several millimeters. However, the best results are achieved with capsules with a size in the range from 300 to 1000 μm. Langer hans islands of humans or mammals typically have a diameter of 50 to 200 [im. Individual cells, such as fibroblasts, leukocytes, lymphoblastoids, pancreatic β-cells, a-cells, S-cells or mixtures thereof in different ratios or other tissue units, can be encapsulated as desired. Microorganisms, including those that have been genetically modified by recombinant DNA or other methods, can also be encapsulated. 25 The continued viability of such living materials depends, among other things, on on the accessibility of the required nutrients, the oxygen transfer, the absence of toxic substances in the medium and the pH of the medium. So far, it has not been possible to obtain such living materials in a physiologically acceptable environment while encapsulating them. The problem has been that the conditions required for membrane formation have been lethal or harmful to the tissue, and prior to the invention of U.S. Patent Application No. 35 No. 24,600, there was no membrane formation process that would allow tissue to survive in healthy condition.

However, it has been found that certain water-soluble substances that are physiologically compatible with living tissue and that can be rendered water-insoluble to form a dimensionally stable, continuous mass can be used to form a "temporary capsule".

or to form a protective barrier around individual cells or groups of cells and that this temporary capsule can be treated to deposit a less temporary semipermeable membrane around the cells without damaging the cells. Such a substance is, typically at a concentration of the order of a few% by weight, added to the culture medium of the tissue, which also contains cells of the inoculum, serum components (if necessary) and, if appropriate, a cellular substrate such as collagen or other high molecular weight water dispersible material that acts as a fixation substrate. When using collagen, the concentration should be in the range of about 10 µg / ml to 1 mg / ml, but is preferably in the order of 100 to 500 (µg / ml.

The solution is then deformed into droplets, which contain the tissue together with its medium, and these 60 are immediately made water-insoluble and gelled, at least in one surface layer. Then the shape-retaining temporary capsules are provided with a less temporary membrane, which in turn can then be selectively destroyed if one wishes to release the tissue without damage. If the material used to form the temporary capsules permits, the interior of the capsule can be liquefied again after the permanent membrane has been formed. This is done by restore

5

654 328

the conditions under which the material is soluble in the medium.

The material used to form the temporary capsules can be any non-toxic, water-soluble material that can be converted to a dimensionally stable mass by changing the ionic environment or concentration. The material should also contain several easily ionizable anionic residues, e.g. Car-boxyl groups, which can react by salt formation with polymers that contain several cationic groups. As will be explained below, the use of a material of this type enables a permanent membrane with a predetermined upper limit of permeability to be deposited without difficulty in the surface layers of the temporary capsule.

The currently preferred materials for forming the temporary capsule are acidic, water soluble, natural or synthetic polysaccharide gums. Such materials are commercially available. They are typically extracted from plant matter and are often used as additives in various foods. Sodium alginate is the currently preferred water-soluble rubber. Alginates with molecular weights in the range above 150,000 u can be used, but because of their molecular dimensions and their viscosity they are usually unable to penetrate the capsule membranes formed in the end. Alginates with lower molecular weights, e.g. 50,000 to 80,000 µ can be more easily removed from the volume within the capsule by diffusion through a membrane with sufficient porosity and are therefore preferred. Other gums that can be used are acidic fractions of guar gum, carrageenan, pectin, tragacanth or xanthan gum.

These materials have glycosidically linked saccharide chains. Their free acid groups are often in the alkali metal salt form, e.g. the sodium form. When a multivalent ion, such as calcium or aluminum, is exchanged for the alkali metal ion, the water-soluble polysaccharide molecules are "crosslinked" to form a water-insoluble, dimensionally stable gel that can be made soluble again by removing these ions by ion exchange or with the aid of a sequestering agent. Although substantially any polyvalent ion capable of forming a salt with the acidic gum is useful, physiologically acceptable ions, e.g. Calcium. This helps to keep the tissue alive. Other multivalent cations can also be used. Magnesium ions do not cause sodium alginate to gel.

A solution with a typical composition comprises equal volumes of a cell culture in its culture medium and a 1 or 2% solution of gum in physiological saline. When using sodium alginate, a 1.2 to 1.6% solution was successfully used. If the cells to be encapsulated are of the type that must be fixed to a fixation substrate to experience mitosis and if the cells are to be grown within the capsules, collagen or other high molecular weight, water-dispersible proteins or polypeptides, either natural or synthetic, can be used , are incorporated into the cell culture and are then enclosed within the volume of the final capsule formed. If a polymer with multiple cationic groups, e.g. Polylysine, used for this purpose, the cationic groups react with anionic groups in the water-soluble rubber to form a substantially water-insoluble matrix which is intertwined with the rubber. The preferred concentrations of such materials are in the order of 100 to 500 (ig / ml of the suspension (including the gum solution).

In the next stage of the encapsulation process, the rubber solution containing the tissue is transferred into droplets of 5 of the desired size. The droplets are then gelled immediately to form dimensionally stable, preferably, but not necessarily, spherical or spheroidal masses. The drop formation can be carried out in a known manner. An example of such a procedure follows.

A tube containing an aqueous solution of polyvalent cations, e.g. a 1.5% calcium chloride solution is provided with a stopper which contains a droplet forming apparatus. The apparatus consists of a housing with an upper air intake nozzle and an elongated hollow body which is fixed in the stopper by frictional force. A 10 ml syringe equipped with a indexing pump is mounted on top of the housing, e.g. a Teflon-coated needle with an inside diameter of 0.25 mm extends through the entire length of the housing. The interior of the housing is designed so that the tip of the needle is exposed to a continuous laminar flow of air that acts as an air knife. In use, the syringe is filled with the solution containing the material to be encapsulated and the indexing pump is actuated to drip droplets of the solution out of the tip of the needle in increments. Each drop is “cut off” by the air flow and falls approximately 2.5 cm down into the calcium chloride solution, where it immediately gels through the absorption of 30 calcium ions. In this case, the distance between the tip of the needle and the surface of the calcium chloride solution is sufficiently large for the sodium alginate cell suspension to take the physically most favorable form, namely a sphere (maximum volume with minimum surface area). The air inside the tube escapes through an opening in the stopper. This leads to the "crosslinking" of the gel and the formation of a highly viscous, dimensionally stable, protective temporary capsule which contains the suspended tissue and its medium. The capsules collect in the solution as a separate phase and can be removed by suction.

In the next stage of the process, a “semi-permeable membrane” is deposited around the surface of the temporary capsules by “crosslinking” surface layers. This can be done by exposing the gelled temporary capsules to an aqueous solution of a polymer containing cationic groups that are reactive with the anionic functional groups of the gel molecules. Polymers containing acid reactive groups such as free imine or amine groups are preferred. In this situation, the polysaccharide rubber is cross-linked by interaction (salt bond formation) between the car-boxyl groups and the amine or imine groups. Permeability can be controlled within certain limits by appropriately selecting the molecular weight of the crosslinking polymer used and by regulating the concentration of the polymer solution and the duration of exposure. A solution of a low molecular weight polymer penetrates further into the temporary capsules within a given time than a high molecular weight polymer. The degree of penetration of the crosslinking agent was related to the resulting permeability. In general, the larger the molecular weight and the lower the penetration, the larger the pore size. In general, polymers with molecular weights in the range of 3,000 to 100,000 µ or more can be used depending on the duration of the reaction, the concentration of the polymer solution and the like

654 328

6

desired degree of permeability. A successful combination of reaction conditions using a polylysine with an average molecular weight of about 35,000 µ comprised the reaction of a physiological saline solution containing 0.0167% poly-lysine with stirring for two minutes. This resulted in membranes with an upper permeability limit of approximately 100,000 u. The optimal reaction conditions suitable for controlling permeability in a given system can easily be determined empirically in view of the above guidelines. Using this method, it is possible to set the upper permeability limit of the membranes to a predetermined value below about 200,000 µ.

Examples of suitable crosslinking polymers are proteins and polypeptides, which can either be natural or synthetic and contain free amino or imino groups, polyethylene amines, polyethylene imines and poly vinyl amines. Polylysine, in both the D and L forms, has been used successfully. Proteins such as polyarginine, polycitrulline or polyornithine can also be used. Polymers with a positive charge density in the higher range, e.g. Polyvinylamine, strongly adhere to the anionic groups of the gel molecules to form stable membranes, but the membranes are rather difficult to destroy.

Treatment with a dilute solution of gum links the free amino groups on the surfaces of the capsules, which could otherwise give the capsules a tendency to clump together.

At this point in the encapsulation process, capsules can be collected that have a semi-permeable membrane that contains a gelled solution of gum, a culture medium compatible with the cell type, cells, and optionally an inner matrix of collagen or other fixation substrate. Since mass transfer within the capsules and through the membranes is to be promoted, the gel is preferably liquefied again to its water-soluble form. This can be done by restoring the conditions under which the rubber is liquid, e.g. by removing the calcium or other multivalent cations from the inner gel. The medium in the capsules can be easily solubilized by immersing the capsule in phosphate buffered saline containing alkali metal ions and hydrogen ions. The monovalent ions exchange for the calcium ions or other multivalent ions within the gum when the capsules are immersed in the solution with stirring. Sodium citrate solutions can be used for the same purpose and serve to sequester the multivalent ions.

Cell cultures that have been encapsulated as described above can be suspended in a culture medium that is specifically designed to meet all the requirements of the cell type in question and are still subject to normal in vitro metabolism. If the culture requires an environment with high molecular components such as serum components, these can be omitted from the medium outside the capsules. Typically, the components that are normally taken up by the cells have a relatively low molecular weight and diffuse easily through the capsule membranes into the micro-milieu of the cells, where they penetrate through the cell membrane. Metabolic products of the cells that are secreted into the medium inside the capsule also diffuse through the capsule membrane and accumulate in the medium outside the capsule if they have a molecular weight below the upper limit of the permeability of the capsule membrane.

The encapsulated cells can be e.g. temperature, pH and ionic milieu, which are the same as in conventional cultures. The products produced by the cells can also be harvested from the medium outside the capsule or from the capsules in a conventional manner. However, the culture method disclosed here has the following advantages:

1. The cells of the culture are protected against shear forces and mechanical damage and against contamination by factors with dimensions above the upper permeability limit of the membranes. This means that the handling and sterility requirements normally associated with breeding methods can be relaxed somewhat, since microorganisms cannot reach the encapsulated cells and cells infected with viruses do not need to infect other cells.

2. The capsules immobilize the cells within a milieu that includes high molecular weight materials, and yet low molecular weight cell nutrients and products are easily removed and introduced. This allows the nutrient medium to be collected and supplemented intermittently or continuously as desired without disturbing the cells.

3. Interesting substances that are generated by the cells are easier to obtain. Eliminated cell products with molecular dimensions that are sufficiently small to penetrate the capsule membranes collect in the medium outside the capsules in a mixture with nutrients. However, high molecular serum components and the like are not released into the medium outside the capsules, which simplifies the extraction of a cell product of interest. Eliminated cell products with molecular dimensions above the upper permeability limit of the membranes accumulate inside the capsules. Of course, cell products that are not excreted through the cell membrane may also be of interest. These can be obtained in a relatively concentrated form by isolating the capsules and then selectively destroying the membranes, e.g. using the following method, and if necessary, destroys the cell membranes.

4. The volume inside the capsules provides an environment that is well suited for cell division. Suspension cultures within the capsules were observed to experience mitosis. Fixation-dependent cells, which grow in normal cultures in a two-dimensional simple layer, multiply with the formation of a considerable amount of cells within the capsule. Such cells use the inner surfaces of the membrane as a substrate and / or attach themselves to the high molecular materials mentioned above and which are arranged inside the capsules. This leads to significant increases in cell density compared to conventional cultures. The continued viability of such grapes is favored by the fact that the surface to volume ratio of the capsules can be quite large, so that all cells have access to the necessary nutrients, oxygen, etc.

In certain situations, it is advantageous to selectively destroy the capsule membranes in order to release the cells without damage. One of the notable examples is the production of interferon. Cells capable of producing interferon must be exposed to certain viruses or nucleic acids in preparation for the interferon generation stage. With multiple interferon

5

10th

IS

20th

25th

30th

35

40

45

50

55

60

65

7

654 328

Induction processes also add reagents to the culture to inhibit protein synthesis. As a result, the growth stage of the breeding process must be carried out under conditions that are quite different from those of the Interfe-ron induction stage. If the substances used for interferon induction have a molecular weight above the upper permeability limit of the capsule membrane (as in the case of virus induction), the induction process cannot be carried out in the encapsulated cell culture. Accordingly, interferon-producing cells, when grown within the capsule, must be released by destroying the membrane in order to subject them to the induction process.

Cells encapsulated in membranes of the type set forth above can be released by a method that uses commercially available reagents, the properties of which do not significantly affect the encapsulated cells. First, the capsules are separated from their suspending medium, washed thoroughly to remove any contaminants present on the exterior of the microcapsules, and then with stirring in a mixed solution of monatomic, polyvalent cations, such as potassium ions, and a destructive polymer with several anionic ones Residues such as a salt of a polysulfonic acid or polyphosphoric acid are dispersed. Heparin, a natural sulfonated polysaccharide, is preferred for this stage. The anionic charge density of the destructive polymer should be equal to or preferably greater than the charge density of the polyanionic material originally used to form the membranes. The molecular weight of the polymer should be at least comparable to the molecular weight of the multi-cationic polymer used to form the membrane, and is preferably larger. Within the suspension of capsules in the mixed solution, the calcium ions compete with the polycationic polymer chains used to form the membrane for anionic groups on the water-soluble rubber. At the same time, the heparin or other polymer with multiple anionic groups dissolved in the solution competes with the rubber in the membrane for the cationic groups on the polymer chains. This results in a water dispersible or preferably water soluble complex of e.g. Polylysine and heparin and to associate the monatomic cations with the molecules of the gel.

This step makes the membrane soluble in the subsequent action of a sequestering agent, which ends the destruction process by taking up monatomic ions from the gel. If fragments of capsule membranes remain in the medium, they can easily be separated from the cells.

The currently preferred solution for the first stage of the selective destruction process contains 1.1% (w / v) calcium chloride and between 500 and 1500 units of heparin per ml of the solution. A volume of microcapsules sufficient to make up between about 20 and 30% of the total volume of the dispersion is added to this solution. Calcium chloride and heparin are preferred for destroying the membranes of capsules containing cells, since both reagents are physiologically compatible with most cells and therefore minimize the possibility of cell damage. Mixtures of aluminum salts or salts of other polyvalent cations (but no magnesium ions) can also be used together with the polysulfonic acid or polyphosphoric acid salts of the type described above.

In general, the concentrations of the monatomic ions and the anionic polymer used in this step can vary within wide limits. The optimal concentrations can easily be determined empirically and depend on the exposure time and the specific polymer used to form the membranes.

The currently preferred sequestering agent for carrying out the selective destruction is sodium citrate, although other alkali metal citrates and alkali metal salts of ethylene-io-diamintetra-acetic acid can also be used. When sodium citrate is used, the optimal concentration is on the order of 55 mmol per liter. It is preferred to dissolve the citrate or other sequestering agent in isotonic saline to minimize cell damage.

The following examples illustrate the invention.

Example 1 Insulin Production 20 islets of Langerhans are obtained from human corpses or animal pancreas and are added to a complete tissue culture (CMRL-1969, Connaught Laboratories, Toronto, Canada) at a concentration of approximately 103 islets per ml. The tissue culture contains all 25 nutrients that are necessary for the continued viability of the islands, as well as the amino acids that are used by the ß-cells to produce insulin. 0.4 ml of a suspension of 103 islands per ml are then added to 0.5 ml of a 1.2% solution of sodium alginate (Sigma 30 Chemical Company) in physiological saline.

A 1.5% calcium chloride solution is then used to gel droplets of the solution in the manner set out above. Droplets with a size 35 order of 300 to 400 µm, which emerge from the tip of the needle, gel immediately upon entering the calcium chloride solution. The gelled capsules are then transferred to a beaker containing 15 ml of a solution which dilutes 1 part of 2% 2- (cyclohexylamino) -ethanesulfonic acid buffer solution in 40 0.6% sodium chloride (isotonic, pH 8.2) with 20 parts of 1% calcium chloride. After immersion for 3 minutes, the capsules are washed twice in 1% calcium chloride.

The capsules are then transferred to 32 ml of a solution containing 1 part 1% polylysine (average molecular weight 35,000 u in 80 parts physiological saline. After 3 minutes the polylysine solution is decanted off. The capsules are filled with 1% calcium chloride washed and optionally resuspended for 3 minutes in such a solution of polyethyleneimine (molecular weight 40,000 to 60,000), which was obtained by diluting a 3.3% polyethyleneimine stock solution in morpholinopropanesulfonic acid buffer (0.2 molar, pH 6) with sufficient l % calcium chloride to give a final polymer concentration of 0.12% 55. The resulting capsules, which have "permanent" semipermeable membranes, are then washed twice with 1% calcium chloride and twice with physiological saline and with 10 ml 0 , 12% alginic acid solution mixed.

60 The capsules don't clump together, and many contain islets of Langerhans. The gel on the inside of the capsules is liquefied again by immersing the capsules in a mixture of saline and citrate buffer (pH 7.4) for 5 minutes. Finally, the capsules are suspended in 65 CMLR-69 medium.

Under the microscope, you can see that these capsules contain a very thin membrane that encloses an island in which individual cells are visible. Molecules with

654 328

8th

a molecular weight up to about 100,000 u can cross the membranes. This enables oxygen, amino acids, nutrients and plasma components used in the culture medium (i.e. low molecular weight components of fetal calf plasma) to reach the island and insulin to be excreted.

After repeated washes with physiological saline, microcapsules obtained by the above procedure and containing approximately 15 islets are suspended in 3 ml of CMRL-1969. When they were 8 days old, the capsules excreted 67 units of insulin per ml in the medium outside the capsule in the presence of 600 mg of glucose per dl in one batch in 1.5 hours. In a second approach, 68 units of insulin per ml were generated in the same time. 1-week-old capsules excreted 25 units of insulin per ml in a first batch in the same medium, but in the presence of 100 mg of glucose per dl, and 10 units of insulin per ml in a second batch.

Example 2

Production of interferon-ß

Human fibroblasts, which were obtained in a known manner by treating human foreskin tissue with trypsin and ethylenediaminetetraacetic acid for 5 minutes at 37 ° C., are suspended in a complete growth medium (CMLR-1969, Connaught Laboratories), which was mixed with 40 vol. -% purified fetal calf serum, 0.8% sodium alginate (Sigma) and 200 µg purified calf skin collagen per ml. The cell density of the suspension is approximately 1.5 × 10 7 cells per ml. Temporary alginate capsules are formed as set out in Example 1. Semipermeable membranes are deposited in surface layers of the capsules by suspending the capsules in a 0.005% (w / v) aqueous solution of polylysine (molecular weight 43,000 u) for 3 minutes.

The resulting capsules are suspended in CMLR-1969 supplemented with 10% fetal calf serum. The above steps are all carried out at 37 ° C. After incubation at the same temperature, the capsules are examined under a microscope to find that they contain fibroblasts that have undergone mitosis within the microcapsules and have a three-dimensional fibroblastic morphology.

After incubation for 4 to 5 days, the encapsulated fibroblasts are subjected to an interferon-ß superinduction method using the Vilcek method. Under a 5% carbon dioxide atmosphere (95% air), the capsule suspension becomes available in the presence of 100 µg poly I-poly C per ml, a double-stranded RNA (known interferon-β-inducer) available from PL Biochemicals, Milwaukee, Wisconsin and incubated 50 jig of cycloheximide per ml (protein synthesis inhibitor, Calbiochem, La Jolla, California) for 1 hour at 37 ° C. After 1 hour the suspended capsules are washed in the medium (CMLR-1969) containing 50 µg cycloheximide per ml and then resuspended in a 5% carbon dioxide atmosphere for 3 hours at 37 ° C in the same solution. At the end of this incubation, the washing step is repeated, whereupon the capsules are resuspended in the medium containing 50 μg cycloheximide per ml and 5 μg actimomycin D per ml (a known RNA synthesis inhibitor, Calbiochem) and below a 5% strength Incubated carbon dioxide atmosphere at 37 ° C for 2 hours. The capsules are then washed twice in the medium and suspended in the serum-free medium at 37 ° C. for 18 to 24 hours; During this time, the interferon-ß fibroblasts, which has a molecular weight of the order of 21,000 u and can be harvested from the medium outside the capsules, are excreted.

Example 3 5 Production of Interferon-β

The procedure of Example 2 is repeated, but the capsule membranes are selectively destroyed by the induction, so that the poly I-poly C has easier access to the fibroblasts. The destruction process is carried out as follows:

Portions of 10 ml of the microcapsule suspension, which contains about 500 to 1000 capsules per ml, are allowed to settle and the suspension medium is suctioned off. The capsules are washed twice with phosphate buffered saline (pH 15 7.4). The washed capsules are then mixed with an aliquot of 3.0 ml of phosphate buffered saline containing 1000 units of heparin per ml and 1.1% (w / v) calcium chloride. The suspension is stirred at 37 ° C for 3 minutes; then the capsules are allowed to settle, the supernatant is suctioned off and the capsules are washed twice with 3.0 ml of 0.15 molar sodium chloride. After the second washing solution has been suctioned off, the capsules are mixed with 2.0 ml of a mixed solution which contains equal amounts of 110 mmol sodium citrate and 25 0.15 molar sodium chloride (pH 7.4). The mixture is swirled by hand for 1 minute to cause the membranes to dissolve, after which the cells are washed twice in the medium.

The cells are then suspended in the medium, subjected to the induction process described in Example 2 and then encapsulated again as described in Example 2. The capsule suspension is then incubated in a serum-free medium for 18 to 24 hours; During this time, interferon-ß is excreted from the cells, 35 penetrates the capsule membranes and collects in the medium outside the capsules

If Examples 2 and 3 are carried out with poly I-poly C (5S) (sedimentation value, poly I and poly C heated to form double-stranded RNA), the following 40 values of the interferon-β production result, expressed in units Interferon-ß / 105 cells:

1.2.

Example 2 25 25

■ »5 Example 3 2500 2500

If Examples 2 and 3 are carried out with poly I-poly C (12S) (sedimentation value, double-stranded as purchased), the following values of the interferon-β-50 production result, expressed in units of interferon-β / 105 cells:

1.2.

Example 2 25 25

Example 3 2500 2500

55

It is believed that the hundredfold increase in the amount produced using the procedure of Example 3 over Example 2 is at least in part due to the fact that the poly I-poly C in the method of Example 3 has better access to the cells Has.

Example 4

The procedure of Example 2 is repeated, but capsules containing no collagen are used. The encapsulated cells were grown in a conventional monolayer culture, treated with trypsin and induction with Poly I-Poly C (5S) while encapsulated in microcapsules. It was found

9

654 328

that the medium present outside the capsules contains 2500 units of interferon-β / 105 cells.

Example 5

Monoclonal antibodies

Hybridoma cells obtained from Herman Eisen at MIT were grown to a density of 3.0 x 106 cells per ml. These cells were fused from mouse spleen cells and mouse myeloma cells in a manner now well known in the art and constituted an immortal cell line that raised antibodies to dinitrophenyl bovine serum albumin in culture. Aliquots of 3 ml of the cell suspension were obtained by adding 2.1 ml of the suspension containing 1.4% sodium alginate to 0.6 ml of fetal calf serum and 0.3 ml of physiological (150 mmolar) saline. Droplets of the suspension were immediately gelled in calcium chloride solution and then treated with a 0.016% by weight solution of poly-L-lysine. The inside of the resulting capsules were then liquefied by immersing them in a solution of 1 part 110 mmolar sodium citrate and 3 parts 150 mmolar saline for 6 minutes. The capsules containing hybridoma cells were then suspended in a mixture of RPMI 1640 medium (Gibco) containing 20% heat inactivated calf serum.

The cell densities of the encapsulated and unencapsulated hybridoma cultures and the amounts of monoclonal antibodies produced by both the encapsulated and the unencapsulated cultures were determined periodically. The results are shown graphically in FIG. 2.

Example 6

Interferon-a from leukocytes

30 ml "buffy coats", obtained from the American Red Cross, were treated with 3.0 ml of 5% ethylenediaminetetra-acetic acid and repeatedly exposed to 0.83% ammonium chloride at 4 ° C for 10 minutes in order to reduce the red blood cells lyse. Centrifugation (1200 rpm at 4 ° C) for 5 minutes between ammonium chloride treatments to separate debris from the remaining intact leukocytes. The cells were then suspended in MEM (minimal essential medium, serum-free - Gibco) diluted by a factor of 100 and stained with tryptan blue for 15 minutes. A cell count performed on a sample showed

that about 1.3 x 109 leukocytes per 30 ml "buffy coat" survived. The cells were then suspended at a density of 1 x 107 cells per ml in the medium supplemented with 2% heat-inactivated fetal calf serum.

Induction was carried out by exposing the cell suspension to sendai viruses (various concentrations in hemagglutination units per ml -Flow Laboratories, Md.) At 37 ° C. with stirring for 1 hour. The virus was then separated from the cells by centrifugation at room temperature and the cells were resuspended in equal volumes of MEM with 4% heat-inactivated fetal calf serum and 1.4% sodium alginate. As stated above, capsules were formed and then resuspended in serum-free and serum-containing media. There were no significant differences in the levels of interferon detected in the off-capsule medium of these test samples. The interferon production values were also the same in comparison cultures that were not encapsulated. The results of these experiments are given below:

Sendai virus

Generated interferon

(HA units / ml)

(Units / 107 cells)

600

10th

300

20th

150

33

75

50

Example 7 io Interferon-a from lymphoblastoids

Namalwa cells from the American Type Culture Collection were grown both in conventional culture and within microcapsules in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum 15. Portions of the cell suspensions were then subjected to interferon induction and production processes, with one portion encapsulated and the other unencapsulated. The cultures contained essentially the same number of cells. 25 mg of bromodeoxyuridine in dop-20 pelt of distilled water per ml was added to both the encapsulated and non-encapsulated cultures to inhibit mitosis. After incubation at 37 ° C for 36 hours, the cells of both cultures were washed and then suspended in RPMI-1640 medium supplemented with 2% heat-inactivated fetal calf serum.

The encapsulated culture was then treated to selectively destroy the capsule membranes. The capsules were washed three times in physiological saline, incubated at 37 ° C for 10 minutes in a solution containing 1000 units of 30 heparin per ml and 1.1% calcium chloride, and then washed again in saline. The washed capsules were then incubated at 37 ° C for 5 minutes with a dilute solution of sodium citrate in physiological saline. By stirring the capsule-35 pension at this point, the membranes dissolve and release the Namalwa cells. The cell suspension is then centrifuged to remove residues and washed several times with citrate / saline solution.

Both cultures were then suspended at a density of 4o 1.0 x 106 cells per ml in fresh RPMI 1640 culture medium supplemented with 2% heat-inactivated fetal calf serum and buffer (pH 7.4).

Then the Bankowski strain of Newcastle Disease Virus in amniotic fluid was added to both the conventional culture and the previously encapsulated culture. The virus was 1.0 x 108 pfu / ml and was purchased from Poultry Health Laboratories, Davis, California. 1 ml of the virus was added per 10 ml of cell suspension. The cultures were incubated at 37 ° C for 24 hours.

The conventional culture was then divided into 5 parts (labeled 1 to 5 below), and the previously encapsulated culture was divided into 4 parts (labeled 6 to 9 below). Each of the 9 aliquots of the culture was then tested for interferon production after the 55 treatments listed below,

1. untreated;

2. resuspended in RPMI 1640 medium with 2% heat inactivated fetal calf serum;

60 3. resuspended in serum-free RPMI-1640 medium;

4. Encapsulated with RPMI 1640 medium and 5% heat inactivated fetal calf serum; Capsules suspended in serum-free medium; 5. Encapsulated with RPMI 160 medium and 5% heat inactivated fetal calf serum; Capsules suspended in medium with 2% fetal calf serum;

654 328

10th

6. suspended again in serum-free medium;

7. Suspended again in medium containing 2% heat-inactivated fetal calf serum;

8. Encapsulated with medium plus 5% heat inactivated fetal calf serum; Capsules suspended in 5 um free medium;

9. Encapsulated with medium plus 5% heat inactivated fetal calf serum; Capsules suspended in medium plus 2% serum.

The following table shows the amount of cells required in 10 of each of cell cultures 1 to 9 to produce one unit of interferon-a:

Cell culture

Amount of cells

Cell culture

Amount of cells

1

30th

6

40

2nd

45

7

40

3rd

-

8th

1000, 360

4th

680

9

200, 100

5

2000

G

2 sheets of drawings

Claims (17)

654 328 1. A method for producing substances produced by living cells, characterized in that A. encapsulates the cells within membranes with a given upper limit of permeability; B. suspending the encapsulated cells in an aqueous culture medium; C. the cells experience metabolism in vitro and excrete the substance; and D. the substance is harvested either from the aqueous medium or from inside the capsules. 2. The method according to claim 1, characterized in that the encapsulation step (A) is carried out by forming the membrane by reaction between cationic groups on polymer chains and anionic groups on a water-soluble rubber to form a water-insoluble, salt-binding matrix. 2nd PATENT CLAIMS 3. The method according to claim 1, characterized in that the encapsulation step (A) is carried out by a) suspending the cells in an aqueous medium which is physiologically compatible with the cells and contains a water-soluble gum with several anionic residues; b) forming droplets containing the cells from the suspension; c) exposing the droplets to a solution of polyvalent, physiologically acceptable cations in order to gel the droplets as discrete, dimensionally stable, water-insoluble temporary capsules; and d) crosslinking surface layers of the temporary capsules to form semipermeable membranes around the droplets by exposing the capsules to a polymer containing several cationic groups that are reactive with anionic residues. 4. The method according to claim 3, characterized in that, in addition, the gel is made soluble again within the membrane produced in step (b). 5. The method according to any one of claims 1 to 4, characterized in that the substance produced has a molecular weight below the predetermined upper permeability limit of the membrane and that the substance is allowed to diffuse through the membranes into the aqueous medium and is harvested therefrom. 6. The method according to any one of claims 1 to 5, characterized in that the cells are encapsulated together with a complete cell culture medium, which is sufficient to maintain the cells and to enable the biosynthesis of the substance in vitro. 7. The method according to any one of claims 1 to 6, characterized in that the aqueous medium used in step (B) is a complete cell culture medium, which is sufficient to maintain the cells and to enable the biosynthesis of the substance in vitro. 8. The method according to any one of claims 1 to 7, wherein the cells require a component with a molecular weight above the upper permeability limit of the membranes, so that the biosynthesis of the substance is possible in vitro, characterized in that in addition this component together with the cells encapsulates. 9. The method according to any one of claims 1 to 8, characterized in that the cells are human or mammalian cells. 10. The method according to any one of claims 1 to 9, characterized in that the cells within the capsule are additionally allowed to experience mitosis. 11. The method according to any one of claims 1 to 10, characterized in that the cells are genetically modified cells. 12. The method according to any one of claims 1 to 11, characterized in that you are in the encapsulation stage (A) produces spheroidal microcapsules with a diameter below 0.5 mm. 13. The method according to any one of claims 1 to 12, characterized in that in step (D) a substance is harvested which is selected from insulin, glucagon, prolactin, somatostatin, thyroxine, steroid hormones, pituitary hormones, interferons, FSH and PTH. 14. The method according to any one of claims 1 to 12, characterized in that in step (D) a substance is harvested which is selected from hormones, interferons, lymphokines and antibodies. 15. The method according to any one of claims 1 to 14, wherein the cells encapsulated in step (A) need contact with a component which has a molecular weight above the upper permeability limit of the membranes in order to maintain the production of the substance, characterized in that encapsulates this component together with the cells in stage (A) and that in stage (B) the aqueous culture medium used is essentially free of this component. 16. The method according to one of claims 1 to 15, characterized in that the predetermined upper limit of permeability is below 1.5 x IO5 u. 17. The method according to any one of claims 1 to 16, characterized in that the cells are hybridoma cells, that the substance produced is monoclonal antibodies with a molecular weight above the predetermined upper limit of the permeability of the membrane and that the antibodies are harvested from inside the capsule.