CH651579A5 - METHOD FOR SELECTIVELY DESTRUCTING A PERMEABLE MEMBRANE. - Google Patents

Description

The invention relates to a method for the selective destruction of a permeable membrane, which has a matrix of a) at least a first polymer with several cationic groups and b) a second polymer with several anionic groups and a first charge density, wherein the first polymer and the second polymer are connected by salt bridges between the anionic and cationic groups, and the use of this method to destroy a permeable membrane that delimits a microcapsule that contains a living cell.

U.S. Patent Application No. 24,600, filed March 28, 1979 by F. Lim, discloses a microencapsulation process that can be used to remove substantially any solid or liquid material within semi-permeable or virtually impermeable capsule membranes encapsulate. An outstanding advantage of this method is that the conditions under which the capsule membranes are formed do not include toxic or denaturing reagents, extreme temperatures or other conditions that damage living cells. The method according to the invention is accordingly well suited for the production of microencapsulated living materials which remain viable and in a healthy state. Because the method allows some degree of control of membrane permeability, it is now possible to encapsulate cell cultures of prokaryotic, eukaryotic or other origin in microcapsules such that the culture cells are protected from contaminating bacteria, high molecular weight immunoglobulins and other potentially harmful factors and remain trapped within a micromilieu that is well suited to their continued viability and ongoing metabolic functions. If the microcapsules are suspended in a conventional culture medium which is sufficient to support the growth of the living cells in question, the cells encapsulated in microcapsules are free to take up substances which are necessary for the metabolism and which diffuse through the membrane and their Eliminate metabolic products through the capsule membrane into the surrounding medium.

The present invention relates to a method for selectively destroying permeable membranes, e.g. can be synthesized during the microencapsulation process set forth in the above-mentioned U.S. patent application without causing any detectable damage to the encapsulated core material. The method according to the invention is applied to membranes which have a water-insoluble matrix which is formed from at least two water-soluble components: at least a first polymer which has a plurality of cationic groups (polycationic polymer, for example polyethylene amine); and a second polymer with multiple anionic groups (polyanionic polymer, e.g., sodium alginate rubber). The two components are by salt5

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bridge between the anionic and cationic groups to form the matrix.

The process according to the invention comprises the following stages: membranes of the type indicated above are exposed to a solution of cations, preferably monatomic or very low molecular weight multivalent cations, and at least one destructive polymer with several anionic groups. These solutions are preferably mixed together. The charge density of the destructive polymer must be sufficient to destroy the salt bridges and should preferably be equal to or greater than the charge density of the polyanionic polymer of the membrane. The solution is brought into contact with the membrane to remove the cations, e.g. Calcium or aluminum, with the polycationic polymer to compete for the anionic groups on the polyanionic polymer. At the same time, the destructive polymer competes with the polyanionic polymer for the cationic groups on the polycationic polymer. This leads to a "softening" or opening of the membranes. To complete the destruction, preferably after a washing operation, the capsules are exposed to a sequestering agent to sequester cations associated with the polyanionic polymer. The preferred sequestering agents are chelating agents such as citrate ions or ethylenediaminetetraacetic acid ions. When the membranes encapsulate viable cells, as in an important application, it is preferred to mix the sequestering agent with an isotonic saline solution.

The currently preferred cation is calcium. Examples of destructive polymers with multiple anionic groups used in the mixed solution include Polysulfonic acid or (preferably) its salts, which can be either natural or synthetic in origin. Excellent results have been obtained using heparin, a natural polymer containing multiple sulfonate groups. Polymers containing poly-phosphoric acid or polyacrylic acid salt groups can also be used. The currently preferred sequestering agent is sodium citrate.

The invention also enables the release of viable cells that are encapsulated in a protective environment. A kind of packaging is thus available that keeps living cell cultures of any origin in a sterile, stabilizing environment, in which they can experience normal metabolism and even mitosis and from which they can then be released.

The invention thus enables living cells encapsulated within permeable membranes to be released by selective destruction of the membranes. The invention further enables the selective destruction of membranes, without damaging adjacent living tissue, and the release of encapsulated, finely divided materials, liquids and solutions.

The method according to the invention for the selective destruction of membranes is applied to membranes which consist of a matrix of polycationic polymers and polyanionic polymers connected by salt bridges. The membranes are usually spheroidal in shape, delimiting an interior containing an encapsulated substance. The method can also be applied to membranes of this type that are not spheroidal in shape.

Although essentially any material (which is compatible with aqueous environments) is encapsulated in liquid or solid form and then by means of the

can be released according to the process, the most important application is currently considered to be the ability to release encapsulated living systems, such as cell cultures. The description is primarily directed to a discussion of cell encapsulation and release. It is readily possible for those skilled in the art to modify the process in connection with the encapsulation of less sensitive materials.

The tissue or cells to be encapsulated can be suspended in an aqueous medium suitable for maintaining and maintaining the ongoing metabolic process of the particular cell type in question. Media suitable for this purpose are well known to those skilled in the art and can often be obtained commercially. The average diameter of the cell mass or other material to be encapsulated can vary widely between a few micrometers and a millimeter or more. For example, Lan gerhans islets from mammals typically have a diameter of 50 to 200 p.m. Tissue fragments and individual cells, the fibroblasts, leukocytes, lymphoblastoids, pancreatic β or a or 5 cells, Langerhans islands, hepatocytes or cells of other tissues can be encapsulated as desired. Microorganisms can also be encapsulated, including those that have been genetically modified by recombinant DNA or otherwise.

The continued viability of such living materials depends, among other things, on the accessibility of the required nutrients, on the oxygen transfer, on the absence of toxic substances in the medium and on the pH of the medium. So far, it has not been possible to keep such a living material in a physiologically acceptable environment while encapsulating it. The problem was that the conditions required for membrane formation were lethal or harmful to the tissue; prior to the invention of the above-referenced U.S. patent application, no method of membrane formation was known to enable the tissue to survive in a healthy condition.

It has now been found that the above-mentioned water-soluble polymers, which are physiologically compatible with living tissue and can be rendered water-insoluble to form a dimensionally stable, coherent mass, for the formation of a "temporary" capsule or protective barrier layer around individual cells, groups of cells or tissues can be used around. Such a substance can, typically in a concentration of the order of 1 to 2% by weight, be added to the culture medium of the tissue. The solution can then be converted into droplets which contain the tissue together with its maintenance or growth medium and can be immediately rendered water-insoluble and gelled, at least in one surface layer. The dimensionally stable temporary capsules can then be provided with a less temporary membrane, which can then be selectively destroyed according to the invention in order to release the encapsulated 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 e.g. by restoring the conditions in the medium under which the material is soluble.

The material used to form the temporary capsules can be any non-toxic, water-soluble material surrounded by a change in the5

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the ionic milieu or the concentration can be converted into a dimensionally stable mass. The material also has several easily ionizable anionic groups, e.g. Carboxyl groups, which are able to react by salt formation with polymers which contain several cationic groups. As explained below, a material of this type enables the deposition of a permanent membrane with a given permeability (including such permeability that the membrane is non-porous for molecular weights on the order of several hundred thousand daltons).

The currently preferred polyanionic material for the formation of the temporary capsule are acidic, water-soluble, natural or synthetic polysaccharide gums. Many 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 anionic polymer. Alginates with molecular weights in the range of more than 150,000 daltons can be used, but because of their molecular dimensions they usually cannot pass through the capsule membranes formed in the end. To capsules. without producing trapped liquid alginate, lower molecular weight alginates, e.g. 40,000 to 80,000 Daltons can be used. The alginate can then be more easily removed from the interior of the capsules by diffusion through a membrane with sufficient porosity. Other polyanionic gums that can be used are acidic fractions of guar gum, carrageenan, pectin, tragacanth or xanthan gum.

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

A solution with a typical composition comprises equal volumes of a cell suspension in its medium and a 1 or 2% solution of gum in physiological saline. When using sodium alginate, a 1.2 to 1.6% solution was used successfully.

In the next stage of the encapsulation process, the gum solution that contains the tissue can be deformed into droplets of a desired size sufficient to encase the cells to be encapsulated. The droplets can then be gelled immediately to form dimensionally stable spherical or spheroidal masses. The drop formation can be carried out in a known manner.

One proceeds e.g. as follows:

A tube containing an aqueous solution of polyvalent cations, e.g. Contains 1.5% calcium chloride solution, is provided with a stopper that contains a drop forming apparatus. The apparatus includes a housing with an upper air intake nozzle and an elongated hollow body which is caused by frictional forces in the

Stopper is attached. A 10 ml syringe, which is equipped with a indexing pump, is mounted on top of the housing, for example, a Teflon-coated needle with an inside diameter of 0.25 mm extends lengthways through the housing. The interior of the housing is designed so that the tip of the needle is exposed to a constant laminar flow of air that acts as an air knife. In use, the syringe is filled with a solution containing the material to be encapsulated and the indexing pump is operated to incrementally force droplets out of the tip of the needle. Each drop is "cut off" by the air flow and falls approximately 2.5 cm into the calcium chloride solution, where it is immediately gelled by absorption of calcium ions. In this case, the distance between the tip of the needle and the surface of the calcium chloride solution is sufficiently large to enable the sodium alginate / cell suspension to assume the physically most favorable shape, namely a spherical shape (maximum volume for minimum surface). The air inside the tube exits 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 accumulate in the solution as a separate phase and are removed by suction.

In the next stage of the encapsulation process, a membrane can be deposited around the surface of the temporary capsules by crosslinking surface layers. This is done by exposing the temporary capsules containing polyanions to an aqueous solution of a polymer containing cationic groups that are reactive with the anionic functional groups in the polyanionic polymer. Polymers containing reactive cationic groups such as free amine groups or combinations of amine and imine groups are preferred. In this situation, the polysaccharide rubber is cross-linked by interaction (salt bond formation) between the carboxyl groups and the amine or imine groups of the polycationic polymer. The permeability can advantageously be controlled within certain limits by selecting the molecular weight of the crosslinking polymer used and varying the exposure time and the concentration of the polymer in the solution. A solution of low molecular weight polymer penetrates further into the temporary capsules than a high molecular weight polymer within a given period of time. The degree of penetration of the crosslinking polymer was related to the resulting permeability. In general, the larger the molecular weight and the less penetration, the larger the pore size. Longer exposure times and more concentrated polymer solutions tend to lower the upper limit of the permeability of the membrane. However, the average molecular weight of the polymer is the most important factor. In general, polymers with a molecular weight in the range of 10,000 to 100,000 Daltons or more can be used depending on the reaction time, the concentration of the polymer solution and the desired degree of permeability. A successful combination of reaction conditions using polylysine with an average molecular weight of approximately 35,000 daltons involved the reaction for 3 minutes while stirring a physiological saline solution containing 0.0167% polylysine. This leads to membranes with an upper permeability limit of approximately 100,000 daltons. In general, higher molecular weight materials form membranes

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which are more difficult to destroy than low molecular weight materials. The charge density of the cross-linking polycationic polymer also influences the pore size and the ease of destruction of the membrane. Generally, higher charge density materials form less porous membranes that are more difficult to disrupt. The optimal reaction conditions suitable for controlling permeability in a given system can easily be determined empirically in light of the above guidelines.

Examples of suitable crosslinking polymers are proteins and polypeptides, which can either be natural or synthetic and have free amino groups or combinations of amino and imino groups, polyethyleneamines, polyethylenimines and polyvinylamines. 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) adhere strongly to the anionic groups of the polyanionic molecules and are more difficult to destroy.

At this point in the encapsulation process, capsules can be collected that have a "permanent" semipermeable membrane that surrounds a gelled solution of gum, culture medium compatible with the cell type, and the cells. If the sole purpose is to preserve the cells in a protective medium, no further steps need to be taken.

However, if the 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 gel. The medium in the capsule can be easily solubilized by immersing the capsules 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 polyvalent ions. Rubber molecules with a molecular weight below the upper limit of the permeability of the membranes can then be removed from the interior of the capsules by diffusion.

Finally, it may be desirable to treat the capsules so that free amino groups or the like, which might otherwise give the capsules a tendency to clump, are linked. This can be done, for example, by immersing the capsules in a dilute solution of sodium alginate.

From the foregoing, it can be seen that no harsh reagents, extreme temperatures or other conditions that are detrimental to the health and viability of the cells need to be used in the membrane formation process. Thus, even sensitive cells such as hepatocytes, leukocytes, fibroblasts, lymphoblasts and cells from various endocrine tissues of humans or mammals can be encapsulated without difficulty. Of course, cells of microbial origin, such as yeast, mold and bacteria, which are better equipped to survive in hostile environments, as well as inert reagents,

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Solids or biologically active materials are encapsulated without damage.

Encapsulated cells of the type described above can be suspended in maintenance media or growth media for storage or cultivation and remain free from bacterial infections. When cells that undergo mitosis in vitro are suspended in growth media, they also experience mitosis in the capsules. Normal in vitro metabolism continues if the factors required for the metabolic processes are so low in molecular weight that they can penetrate the capsule membrane or if they are encapsulated together with the cells. Metabolic products of the cells penetrate the membrane and accumulate in the medium if they have a molecular weight below the upper limit of permeability. The cells can be stored, transported or grown in encapsulated form as desired and can be released from their protective environment without damage using the method described below to selectively destroy the membranes.

According to the invention, the encapsulated material is released by a two-step process using commercially available reagents with properties that do not adversely affect the encapsulated cells.

One proceeds e.g. as follows:

First, the capsules are separated from their suspending medium, washed thoroughly to remove any contaminants, and then with stirring in separate solutions or, preferably, in a mixed solution of cations such as calcium ions or other monatomic or low molecular weight cations and a destructive polymer dispersed several anionic residues, such as polysulfonic acid groups. Polymers containing polyphosphoric acid or polyacrylic acid residues can also be used. Heparin, a natural, sulfonated polysaccharide, is preferred for destroying the membranes containing cells. The charge density of the destructive polymer must be sufficient to destroy the salt bridges. Thus, the anionic charge density can be equal to, or preferably greater than, the charge density of the inner polyanionic polymer (e.g., the rubber) that was originally used to form the membranes. The molecular weight of the destructive polymer should be at least comparable to, and preferably greater than, the molecular weight of the inner polycationic polymer that was used in forming the membrane. Within the suspension, the calcium ions compete with the inner polycationic polymer that forms the membrane for anionic groups on the polyanionic polymer. At the same time, the destructive polymer dissolved in the solution competes with the polyanionic rubber in the membrane for cationic groups on the polycationic polymer. This leads to a water-dispersible or preferably water-soluble complex of, for example, polylysine and the polyanionic polymer and to association of the cations with the gel molecules.

This operation makes the membrane suitable for the subsequent action of a sequestering agent, which brings the destruction process to an end by sequestering 2- or 3-valent ions from the gel. Typically, capsule membrane residues remaining in the medium, if present, can be easily separated from the cells.

The currently preferred solution for the first stage of the selective destruction process comprises 1.1% (w / w

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Vol.) Calcium chloride and 500 to 1500 units of heparin per ml of solution. A volume of microcapsules sufficient to form between about 20 and 30% of the total volume of the suspension is added to this solution. Calcium chloride and heparin are preferred when membranes of capsules containing cells are destroyed because both reagents are physiologically compatible with most cells and minimize the possibility of cell damage. Mixtures of aluminum salts and salts of other polyvalent cations (but no magnesium ions) can also be used together with the polysulfonic acid or other acid salt of the type discussed above.

In general, the concentrations of ions and anionic polymer in the solution used in this step can vary within wide limits. The optimal concentrations can easily be determined empirically. The lowest effective concentration for a particular batch of encapsulated cells is preferred.

The currently preferred sequestering agent for carrying out the selective destruction is sodium citrate, although other alkali metal citrates and alkali metal salts of ethylenediaminetetraacetic acid can also be used. When sodium citrate is used, the optimal concentration is on the order of 55 mmol per liter. If the capsule membranes that are destroyed contain viable tissue, the citrate is preferably dissolved in isotonic saline to minimize cell damage.

Examples 5 to 8 illustrate the invention. Examples 1 to 4 relate to the capsule formation not claimed by the invention and Examples 5 to 8 relate to the destruction of the membranes according to the invention.

Example 1 Encapsulation of Pancreatic Tissue

Langerhans islets are obtained from rat pancreas and added at a concentration of approximately 103 islets per milliliter to a complete tissue culture medium (CMRL-1969, Connaught Laboratories, Toronto, Canada). The tissue culture contains all the nutrients necessary for the continued viability of the islands, as well as the amino acids that are used by the cells to produce hormones. 0.4 ml of an island suspension containing approximately 103 islands per milliliter is then added to 0.5 ml of a 1.2% solution of sodium alginate (Sigma Chemical Company) in physiological saline.

Then a 1.5% calcium chloride solution is used to gel droplets with a diameter of the order of 300 to 400 µm. After the supernatant solution has been removed by suction, the gelled droplets are transferred to a beaker containing 15 ml of a solution containing 1 part of a 2% 2- (cyclohexylamino) -ethanesulfonic acid buffer solution in 0.6% saline solution ( isotonic, pH = 8.2), diluted with 20 parts of 1% calcium chloride. After immersion for 3 minutes, the capsules are washed twice with 1% calcium chloride.

The capsules are then transferred to 32 ml of a solution of 1 part of 1% polylysine (average molecular weight 35,000 atomic mass units) in 80 parts of physiological saline. After 3 minutes, the polylysine solution is decanted. The capsules are washed with 1% calcium chloride and if necessary again for 3 minutes in a solution of polyethylene

imine (molecular weight 40,000 to 60,000) suspended by diluting a 3.3% polyethyleneimine stock solution in morpholinopropanesulfonic acid buffer (0.2 molar, pH = 6) with so much 1% calcium chloride that 5 has a final polymer concentration of 0 , Results in 12%. The resulting capsules with "permanent" semipermeable membranes are then washed twice with 1% calcium chloride and twice with physiological saline and mixed with 10 ml 0.12% io alginic acid solution.

The capsules do not clump together, and many can be seen to contain Langerhans Islands. The gel on the inside of the capsules is liquefied again by immersing the capsules in a Geis mixture of saline and citrate buffer (pH = 7.4) for 5 minutes. Finally, the capsules are immersed in the CMLR-69 medium.

Under the microscope, it is found that the capsules have a thin membrane that encloses an island 20 in which individual cells are visible. Molecules with a molecular weight of up to approx. 100,000 can cross the membranes. This allows oxygen, amino acids, nutrients and plasma components used in culture media (e.g. the low molecular weight components of fetal calf serum) to reach the island and insulin to be excreted.

Example 2 Encapsulation of Hepatocytes

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The procedure of Example 1 is repeated, but using 0.5 ml of a liver cell suspension containing approx. 105 cells per ml instead of the 0.4 ml island suspension. The continued viability of liver 35 cells was demonstrated by the dye exclusion method (trypan blue exclusion) and by their observed ability to continuously produce urea. It is known that liver tissue can absorb toxins from its environment in vitro. Accordingly, 40 toxins, the molecular weight of which is so low that they pass through the semipermeable membranes,

detoxified by the cells.

Example 3

45 Encapsulation of activated carbon

The procedure of Example 1 is repeated, but finely divided activated carbon is suspended directly in the sodium alginate solution, the half ml of tissue suspension is omitted and polylysine with an average molecular weight of 35,000 is used as the crosslinking agent. As long as the carbon particles are smaller than the smallest inside diameter of the capillary used to generate the droplets, activated carbon with a high specific surface area, which is surrounded by a semipermeable membrane, is obtained. These membranes effectively prevent the escape of activated carbon pieces or dust, but can still be used to absorb materials of any given molecular weight range from liquid passed through the capsules.

Example 4

Encapsulation of human fibroblasts

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Human fibroblasts obtained by treating human foreskin tissue with trypsin and ethylenediaminetetraacetic acid for 5 minutes

37 ° C. in a known manner are suspended in a complete growth medium (CMLR-1969, Connaught Laboratories), supplemented with 40% by volume of purified fetal calf serum, 0.8% sodium alginate (Sigma) and 200 mg / ml of purified calf skin collagen. The cell density of the suspension is approximately 1.5 × 10 7 cells per ml. Temporary alginate capsules are formed as indicated above. Semipermeable membranes are deposited in surface layers of the capsules by suspending the capsules in a 0.005% (w / v) aqueous solution of poly-L-Ly-sin (molecular weight 43,000 Daltons) 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, when the capsules are examined under a microscope, they are found to contain fibroplasts that have undergone mitosis within the microcapsules and show a three-dimensional fibroblastic morphology.

Example 5

Microcapsules from one of Examples 1 to 4 can be treated as follows in order to selectively destroy the capsule membranes without damaging the encapsulated core material.

Portions of 10 ml of the microcapsule suspensions containing approximately 500 to 5000 capsules per ml are allowed

settle and sucks off the suspension medium. The capsules are washed twice with saline. The washed capsules are then mixed with an aliquot of 3.0 ml of saline containing heparin in various concentrations, indicated below, and 1.1% (w / v) calcium chloride. After completing this stage, capsules containing alginate will show a gelled, dimensionally stable inner core. The suspension is stirred at 37 ° C. for 10 minutes, after which 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 wash solution has been suctioned off, the capsules are mixed with 2.0 ml of a mixed solution which contain the same volume of 110 mmol sodium citrate and 0.15 mol sodium chloride (pH = 7.4).

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Capsule membranes treated with 1000 units of heparin per ml and swirled in the sodium chloride-sodium citrate solution for 1 minute were completely disintegrated. The same result is achieved with capsules which have been treated with 2,000 units of heparin per ml for 2 minutes and then swirled by hand for 15 to 30 seconds. Lower heparin concentrations are preferred because the possibility of cell damage decreases.

After the membranes have dissolved, any existing membrane fragments can be removed by suction and washing. After the released cells have been resuspended in culture medium, they can be tested using the tryptan blue exclusion method, which is found to be in a healthy, viable state with relatively few cells showing dye uptake.

Example 6

After washing, capsules produced according to Example 3 are treated with 3.0 ml of a solution containing 1000 units of heparin per ml and 1.0% aluminum chloride with stirring for 6 minutes. After the supernatant has been aspirated, the core material is released by swirling the capsules with a 0.1 molar solution of sodium citrate for 30 to 90 seconds.

Example 7

The procedure of Example 6 is repeated, except that 0.10 molar sodium ethylenediaminetetraacetic acid sodium salt with a pH of 7.0 is used instead of the sodium citrate, resulting in rapid destruction of the capsule membranes.

Example 8

Capsules made according to Example 3 are washed after washing with 3.0 ml of an aqueous solution containing 10 mg of polyvinyl sulfate (molecular weight approximately 50,000 Daltons) per ml and 1% calcium chloride. The aftertreatment with 0.10 molar sodium citrate leads to a practically complete dissolution of the capsules.

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Claims (10)

651579 1. A method of selectively destroying a permeable membrane having a matrix of at least a first polymer with a plurality of cationic groups and a second polymer with a plurality of anionic groups and a first charge density, the first polymer and the second polymer being bridged between the anionic and cationic groups, characterized in that one A. exposing the membrane to a solution of cations and at least one destructive polymer with several anionic groups, the destructive polymer having a charge density sufficient to destroy the salt bridges; B. allows the cations to compete with the first polymer for the anionic groups on the second polymer and the destructive polymer with the second polymer to compete for the cationic groups on the first polymer; and C. Sequenced cations associated with the second polymer after step B. 2. The method according to claim 1, characterized in that one carries out the sequestering step (C) by exposing the membranes to a solution which contains a chelating agent, which is preferably selected from citrate ions and ethylenediaminetetraacetic acid ions. 2nd PATENT CLAIMS 3. The method according to claim 1 or 2, characterized in that the first polymer contains several amino groups. 4. The method according to any one of claims 1 to 3, characterized in that the second polymer is an acidic water-soluble rubber, preferably an alginate. 5. The method according to any one of claims 1 to 4, characterized in that heparin is used as the destructive polymer and calcium ions are used as cations. 6. The method according to any one of claims 1 to 5, characterized in that the first polymer is selected from group a) proteins which have several amino acid units with free amino groups; b) proteins which have several amino acid units with free imino groups; c) polypeptides which have several amino acid units with free amino groups; d) polypeptides which have several amino acid units with free imino groups; e) polyvinylamines; f) polyethyleneimines; g) polyethylene amines; and h) mixtures thereof; is. 7. The method according to any one of claims 1 to 6, characterized in that one selects the destructive polymer from a) polysulfonic acids; b) polyphosphoric acids; c) salts thereof, preferably polysulfonic acid salts; and d) mixtures thereof. 8. The method according to any one of claims 1 to 7, characterized in that the charge density of the destructive polymer having a plurality of anionic groups is greater than the first charge density. 9. Application of the method according to any one of claims 1 to 8 for destroying a permeable membrane which delimits a microcapsule which contains a living cell. 10. Use according to claim 9, characterized in that the first polymer is a protein and the second polymer is a water-soluble rubber, in particular a sodium alginate, and that calcium ions are used as cations and heparin is used as the destructive polymer with several anionic groups, wherein the sequestering step is preferably carried out with a solution of citrate in a solution which is physiologically compatible with the cell.