SE454181B - SET TO SPLET SALT BRIDGES IN CAPSEL MEMBRANE FOR RELEASEING THE CONTENT, SEPARATELY LIVE CELLS - Google Patents

Description

To support the growth of the cells in question, the microencapsulated cells are free to ingest substances required for metabolism and diffuse through the membrane and to secrete their metabolic products through the capsule membrane into the surrounding medium.

Summary of the Invention The present invention is directed to a method of selectively disintegrating certain membranes synthesized during the microencapsulation procedure set forth in the above application, without any detectable damage to the encapsulated core material. More specifically, the method of the invention is applied to membranes which comprise a water-insoluble matrix formed from at least two water-soluble components: a polymer which includes multiple cationic moieties (polycationic polymer, eg polyethylene amine); and a polymer with multiple anionic units (polyanionic polymer, eg sodium alginate rubber). The two components are connected to each other by salt bridges between the anionic and cationic units to form the matrix.

The method of the invention comprises the steps of exposure to membrane: of the type indicated above for a solution of cations, preferably monoatomic or very low molecular weight multivalent cations, and a solution of a stripping polymer with a plurality of anionic units. Preferably, these solutions are mixed together. The anionic charge of the polymer should be sufficient to separate 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 contacted with the capsules to allow the cations, eg calcium or aluminum, to compete with the polycationic polymer in the capsule membrane for anionic sites on the polyanionic polymer. At the same time, the stripping polymer, which has a plurality of anionic units, competes with the polyanionic polymer for cation sites on the polymer chains. This results in "weakening" or "unbundling" of the capsule membranes. To complete the separation, the capsules are washed and then exposed to a sequestering agent to remove cations associated with the polyanionic polymer. The preferred sequestering agent is a chelating agent or ED citrate ions. If, as is the case in an important embodiment, the capsules contain viable cells, it is preferred to mix the sequestering agent with an isotonic saline solution.

The presently preferred cation is caclium.

Examples of the stripping polymer having a plurality of anionic groups used in the blended solution include polysulfonic acids or (preferably) their salts, either natural or synthetic. Outstanding results have been obtained using heparin, a natural polymer containing a number of sulfonate groups. Polymers which contain polyphosphoric acid or polyacrylic acid salt units can also be used. The presently preferred sequestering agent is sodium citrate.

The invention also relates to a method of encapsulating viable cells within a protective environment and of later releasing the cells. Thus, the invention provides what can be described as a package that keeps alive cell cultures of any origin in a sterile, stabilizing environment, in which they can undergo normal metabolism and even mitosis and from which the latter can be released.

Accordingly, it is an object of the present invention to provide a method of encapsulating living cells within permeable membranes and later selectively dissociating the membranes to release the cells.

Another object is to provide a method of selectively disrupting membranes. Another object of the invention is to disintegrate the membranes without damaging nearby living tissue. Yet another object of the invention is to provide a method of encapsulating and later releasing atomized materials, liquids and solutions.

These and other objects and features of the invention will become apparent from the following description of certain important embodiments.

Description The selective membrane modification method according to the invention is practiced on membranes consisting of a salt bridge-bonded matrix of a polycationic polymer and a polyanionic polymer. Usually the membranes have a spheroidal shape which defines an enclosed interior, which contains an encapsulated substance. However, the method can also be practiced on membranes of this type which have another even spheroidal shape.

Although substantially any material (compatible with aqueous environments) in liquid or solid form can be encapsulated and later released without damage by the method of the present invention, its presently most significant use lies in its ability to encapsulate and later release - living systems, such as cell cultures. Accordingly, the following description will be mainly about encapsulation and release of cells. Those skilled in the art can easily adapt the method according to the invention to encapsulation of less delicate materials.

Encapsulation The tissue or cells to be encapsulated are suspended in an aqueous medium, which is suitable for maintaining and supporting the ongoing metabolic processes of the particular cell type in question. Media suitable for this purpose are well known to those skilled in the art and are often commercially available. The average diameter of the cell mass or any other material to be encapsulated can vary widely from a few um to 1 mm or more. Langerhans cell islands from mammals, for example, typically have a diameter of 50 to 200 μm. Tissue fragments and individual cells, such as fibroblasts, leukocytes, lymphoblastoids, pancreatic beta, alpha or delta cells, Langerhans islets, hepatocytes or cells of other tissue can be encapsulated if desired. Microorganisms can also be encapsulated, including those that have been genetically modified by recombined DNA or other techniques.

The ongoing viability of such living material depends, among other things, on the availability of required nutrients, oxygen transfer, the absence of toxic substances in the medium and the pH of the medium. Therefore, it has not been possible to maintain such living material in a physiologically compatible environment during simultaneous encapsulation. The problem has been that the conditions required for membrane formation have been lethal or harmful to the tissue, and prior to the invention according to the above application there has been no method of membrane formation which has allowed tissue to survive in a state of good health.

However, it has been discovered that certain water-soluble substances, which are physiologically compatible with living tissue and which can be made water-insoluble to form a shape-retaining, cohesive mass, can be used to form a "temporary capsule" or protective barrier layer around individual cells. groups of cells or tissues. Such a substance is typically added at a concentration of the order of 1-2% by weight to the tissue culture medium. The solution is then formed into droplets containing the tissue together with its maintenance or culture medium, and immediately made water-insoluble and gelled, At least in a surface layer, then the shape-retaining temporary capsules are provided with a more permanent membrane, which in accordance with the present invention can later be selectively disintegrated to release the encapsulated tissue without damage. used to form the temporary capsules allows, the capsule contents can be reclaimed tskas after the formation of the permanent membrane. This is done by re-establishing the conditions in the medium under which the material is soluble.

The material used to form the temporary capsules can be any nontoxic, water-soluble material, which can be converted into a shape-retaining mass by changing the ambient ion environment or cell concentration. The material also comprises a plurality of, easily ionized, anionic units, for example carboxyl groups, which can react by salt bond formation with polymers which contain a plurality of cationic groups. As will be explained below, this type of material allows deposition of the permanent membrane with a selected permeability (including substantially non-porous to a level of several hundred thousand daltons).

The presently preferred polyanionic materials for the formation of the temporary capsule are acidic, water-soluble, natural or synthetic polysaccharide rubbers. Many such materials are commercially available. They are typically extracted from vegetable materials and are often used as additives to various foods. Sodium alginate is the presently preferred anionic polymer. Alginate in the molecular weight range of 150 000 + daltons can be used, but due to its molecular dimensions will usually lack the ability to cross the finally formed capsule membranes. To make capsules without entrapped, liquid alginate, alginate with a lower molecular weight, u 10 15 20 25 30 454 181 7 eg 40,000 - 80,000 daltons, can be used. In the finished capsule, the alginate can be more easily removed from the intracapsular volume by diffusion through a membrane with sufficient porosity. Other useful polyanionic rubbers include acidic fractions of guar gum, carrageenan, pectin, tragacanth or xanthan gum.

These materials include glycoside-linked saccharide chains. Their free acid groups are often present in alkali metal ion form, eg sodium form. If a multivalent ion, such as calcium, strontium or aluminum, is exchanged for the alkali metal ion, the liquid, water-soluble polysaccharide molecules are "crosslinked" to form a water-insoluble, shape-preserving gel, which can be redissolved upon removal of the ions by ion exchange or via a Although substantially any multivalent ion that can form a salt with the acidic gum is functional, it is preferred to use physiologically compatible ions, such as calcium.

This tends to preserve the tissue in a living state. Other multivalent cations can be used for less delicate materials. Magnesium ions are ineffective in gelling sodium alginate.

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

In the next step of the encapsulation process, the rubber solution, which contains the tissue, is formed into droplets of a desired size sufficient to enclose the cells to be encapsulated. Thereafter, the droplets are immediately gelled to form shape-retaining spherical or spheroidal masses. The droplet forming can be performed by known techniques.

A tube containing an aqueous solution of multivalent cations, eg 1.5% CaCl2 solution, is provided with a stopper which holds a droplet forming apparatus. This apparatus comprises a housing with an upper air intake nozzle and an elongate hollow body which is frictionally adapted to the plug. A 10 cm syringe, which is equipped with a step pump, is mounted on top of the housing with, for example, a 0.025 cm inner diameter Teflon-coated needle that passes through the length of the housing. The interior of the housing is designed so that the tip of the needle is exposed to a constant laminar air flow, which acts as an air knife. In use, with the syringe full of solution containing the material to be encapsulated, the step pump is operated to force droppings of solution from the needle tip in increments. Each drop is "cut off" by the air stream and falls about 2.5 cm into the CaCl2 solution, where it immediately gels by absorption of calcium ions. The distance between the tip of the needle and the surface of the CaCl 2 solution is large enough, in this case, to allow the sodium alginate / cell suspension to take the most physically favorable form; a sphere (maximum volume for minimum surface area). Air inside the pipe leaks through an opening in the plug. betta results in "crosslinking" of the gel and in the formation of a highly viscous, shape-preserving, protective temporary capsule, which contains the suspended tissue in its medium. The capsules are collected in the solution as a separate phase and separated by aspiration.

In the next step of the method, a membrane is deposited around the surface of the temporary capsules by crosslinking the surface layer. This is done by subjecting the temporary capsules comprising polyanion to an aqueous solution of a polymer containing cationic groups which are reactive with anionic functionalities in the polyanionic polymer. Polymers which contain reactive cationic groups, such as free amine groups, or combinations of amine and imine groups, are preferred. In this situation, the polysaccharide gum is crosslinked by interaction (salt bond formation) between the carboxyl groups and the amine or imine groups of the polycationic polymer. Advantageously, the permeability can be controlled within certain limits by selecting the molecular weight of the crosslinking polymer used and by varying the exposure time and the concentration of polymer solution. A solution of a low molecular weight polymer penetrates, for a given period of time, further into the temporary capsules than a high molecular weight polymer. The degree of penetration of the crosslinking agent has been correlated with the resulting permeability. In general, the higher the molecular weight and less penetration, the larger the pore size. Longer exposures and more concentrated polymer solutions tend to reduce the upper permeability limit of the resulting membrane. However, the average molecular weight of the polymer is the dominant factor. In general, polymers in the molecular weight range lo ooo to ioo ooo daltons or larger can be used, depending on the duration of the reaction, the concentration of the polymer solution and the desired degree of permeability. A batch of successful reaction conditions, using lysine having an average molecular weight of about 35,000 daltons, involved reacting for 3 minutes, with stirring, a physiological saline solution containing 0.016? % lysine. This results in membranes with an upper permeability limit of about 100,000 daltons. In general, high molecular weight materials form membranes that are more difficult to disintegrate compared to low molecular weight materials. The charge density of the crosslinking polycationic polymer also affects the pore size and the ease with which the membrane can be broken.

In general, high charge density materials form less porous membranes, which are more difficult to disintegrate. Optimal reaction conditions suitable for regulating permeability in a given system can be easily determined empirically by means of the foregoing guidance.

Examples of suitable crosslinking polymers include proteins and polypeptides, either natural or synthetic, with free amino groups or combinations of amino and imino groups, polyethyleneamines, polyethyleneimines and polyvinylamines. Polylysine, in both the D and L forms, has been used successfully. Proteins such as polyarginine, polycitrulline or polyornithine are also useful. Polymers in the higher range of positive charge density (eg polyvinylamine) adhere strongly to the anionic groups of the polyanionic molecules and are more difficult to separate.

At this stage of encapsulation, capsules comprising a "permanent" semipermeable membrane around a gelled gum solution, cell type compatible culture medium and the cells can be collected. If the purpose is simply to preserve the cells in a protective environment, no further steps need to be performed. However, if mass transfer is to be promoted within the capsules and through the membranes, it is preferable to re-liquefy the gel to its water-soluble form. This can be done by restoring the conditions under which the rubber is in liquid form, for example by removing calcium ions or other multifunctional cat ions from the gel. The medium in the capsule can be redissolved simply by immersing the capsules in phosphate buffered saline, which contains alkali metal ions and hydrogen ions. Monovalent ions exchange with calcium ions or other multifunctional ions within the rubber when the capsules are immersed in the solution while stirring. Sodium citrate solutions can be used for the same purpose and serve to separate the divalent ions. Rubber molecules with a molecular weight below the upper permeability limit of the membranes can then be removed from the intracapsular volume by diffusion.

I »10 15 20 25 30 35 454 181 ll Finally, it may be desirable to treat the capsules to bind free amino groups of the type which may otherwise give the capsules a tendency to clump together.

This can be done, for example, by immersing the capsules in a dilute solution of sodium alginate.

From the foregoing it appears that no strong reagents, extreme temperatures or other conditions which are harmful to the health and viability of the cells need be used in the membrane formation method. Thus, even sensitive cells, such as mammalian hepatocytes, leukocytes, fibroblasts, lymphoblasts and cells from various endocrine tissues can be encapsulated without difficulty. Of course, cells of microbial origin, such as yeast, mold and bacteria, which are better adapted to survive in hostile environments, as well as inert reagents, solids, or biologically active materials, can also be encapsulated without damage.

Encapsulated cells of the type described above can be suspended in maintenance medium or culture medium for storage or culture and will remain free of bacterial infection. If suspended in culture medium, cells undergoing mitosis in vitro will do so within the capsules. Normal in vitro metabolism continues, provided that the factors required for metabolic processes have a sufficiently low molecular weight so that they can penetrate the capsule membrane, or are encapsulated together with the cells. The cells' metabolic products (if they have a molecular weight below the upper permeability limit) penetrate the membrane and accumulate in the medium. The cells can be stored, transported or cultured in encapsulated form as desired, and can be released from their protective environment without damage by the following method of selectively disintegrating the membranes.

Membrane Disruption In accordance with the invention, the encapsulated material can be released by a two-step method, which involves commercially available reagents with properties that do not adversely affect the encapsulated cells.

First, the capsules are separated from their suspension medium, washed thoroughly to remove all contaminants and then dispersed, with stirring, in a separate or preferably mixed solution of cations, such as calcium ions or other monoatomic (low value multivalent cation). molecular weight) and a stripping polymer having a plurality of anionic moieties, such as polysulfonic acid groups. Polymers containing “phosphoric acid or polyacrylic acid units can also be used.

Heparin, a natural sulfonated polysaccharide, is preferred for the breakdown of membranes that contain cells.

The anionic charge of the stripping polymer used must be sufficient to separate the salt bridges.

Thus, the anionic charge density may be equal to or preferably greater than the charge density of the inner polyanionic polymer (eg, the rubber) originally used to form the membranes.

The molecular weight of the stripping polymer should be at least comparable to and preferably greater than the molecular weight of the inner polycationic polymer used to form the membrane. Within the suspension, the calcium ions compete with the inner polycationic polymer, which comprises the membrane, for anionic sites on the polyanionic polymer. At the same time, the stripping polymer, which is dissolved in the solution, competes with the polyanionic rubber in the membrane for cationic seats on the polycationic polymer. This results in a water-dispersible or preferably water-soluble complex of, for example, polylysine and the polyanionic polymer, and in association of the cations with gel molecules.

This step makes the membrane susceptible to subsequent exposure to a sequestering agent, which completes the separation process by taking up di- or trivalent ions from the gel. Typically, capsule membranes, if present, which remain in the medium can be easily separated from the cells.

The presently preferred solution for the first step of the selective filtration process comprises 1.1% calcium chloride (w / v) and between 500 to 1500 units of heparin per ml of solution.

A volume of microcapsules is added to this solution which is sufficient to constitute between about 20% and 30% of the total volume of the suspension. Calcium chloride and heparin are preferred when dissociating membranes of cell- * containing capsules, as both reagents are physiologically compatible with most cells and minimize the possibility of cell damage. Mixtures of aluminum salts or other multivalent cations (but not Mg ++ ions) may also be used with the polysulfonic acid salt or other acid salt of the type indicated above.

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

The presently preferred sequestering agent for performing the selective decomposition is sodium citrate, although other alkali metal citrate salts and alkali metal EDTA may also be used. When sodium citrate is used, the optimum concentration is in the order of 55 mM. When the disassembled capsule membranes contain viable tissue, it is preferred that the citrate be dissolved in isotonic saline, to minimize cell damage.

The invention will be further understood from the following non-limiting examples.

Capsule formation EXAMPLE 1: Encapsulation of pancreatic tissue Langerhans islets are obtained from rat pancreas and added to a complete tissue culture (CMRL 10 l5 20 25 30 35 454 181 14 -1969 Connaught Laboratories, Toronto, Canada) at a concentration of about 103 cell islands per milliliter. The tissue culture contains all the nutrients required for continued viability of the islets as well as the amino acids used by the cells to produce hormones. Four tenths of a milliliter of a cellulose suspension containing about 103 is then added to half a milliliter of 1.2% sodium alginate (Sigma Chemical Company) in physiological saline. - Then a 1.5% calcium chloride solution was used to gel drops in the order of 300-400 μm in diameter. After removal of the supernatant solution by aspiration, the gelled droplets are transferred to a beaker containing 15 ml of a solution comprising a portion of a 2% 2- (cyclohexylamino) ethanesulfonic acid buffer solution in 0.6% NaCl (isotonic, pH = 8.2) diluted with 20 parts 1% CaCl 2. After 3 minutes of immersion, the capsules are washed twice in 1% CaCl2. islets per milliliter The capsules are then transferred to a 32 ml solution containing l / 80 of 1% polylysine (average molecular weight 35,000 amu) in physiological saline. After 3 minutes, the polylysine solution is decanted. The capsules are washed with 1% CaCl 2 and optionally resuspended for 3 minutes in a solution of polyethyleneimine (molecular weight 40,000-60,000) which is prepared by diluting a 3.3% stock solution of polyethyleneimine in morpholinopropanesulfonic acid buffer (0.2M, pH = 6) with sufficient 1% CaCl2 to give a final polymer concentration of 0.12%. The resulting capsules, with "permanent" semipermeable membranes, are then washed twice with 1% CaCl2, twice with physiological saline and mixed with 10 ml of 0.12% alginic acid solution.

The capsules resist clumping and can be seen to contain Langerhan's islets. Gel on the inside of the capsules is re-liquefied by immersing the capsules in a mixture of saline and citrate buffer (pH 7.4) for 5 minutes.

Finally, the capsules are suspended in CMRL-69 medium.

Under a microscope, these capsules are observed to comprise a thin membrane, which surrounds a cell island within which individual cells can be seen. Molecules with a molecular weight of up to about 100,000 can cross the membranes.

This allows oxygen, amino acids, nutrients and plasma components used in low molecular weight culture media (eg fetal calf serum components) to reach the cell islands and allows insulin to be secreted. EXAMPLE 2: Encapsulation of hepatocytes The procedure of Example 1 was repeated except that 0.5 ml of a liver cell suspension, containing about 105 cells per milliliter, was used instead of the 0.4 ml cell suspension. The ongoing viability of the liver cells was demonstrated by the color exclusion technique (tryptane blue exclusion) and by their observed ability to continuously produce urea. It is known that liver tissue, in vitro, can absorb toxins from its environment. Consequently, the cells detoxify toxins with a molecular weight low enough to pass through the semipermeable membranes.

EXAMPLE 3: Encapsulation of Activated Carbon The procedure of Example 1 is repeated except that particulate activated carbon is suspended directly in the sodium alginate solution, half a millimeter of tissue suspension is excluded and polylysine having an average molecular weight of 35,000 is used as a crosslinking agent. As long as the carbon particles are smaller than the smallest inner diameter of the capillary used to generate the droplets, high surface area carbon surrounded by a semipermeable membrane is obtained. These effectively prevent the escape of carbon shavings or coal dust, and can still be used to absorb materials with any pre-selected molecular weight range from fluid passing through the canisters. EXAMPLE 4: Encapsulation of human fibroblasts Human fibroblasts, obtained by treating human foreskin tissue with trypsin and EDTA for 5 minutes at 37 ° C in a known manner, are suspended in a complete culture medium (CMLR 1969). , Connaught Laboratories) increased by 40% (v / v) of purified calf fetal serum, 0.8% sodium alginate (Sigma) and 200 mg / ml purified calfskin collagen. The density of the cell suspension is about 1.5 x 10 6 cells / ml. The temporary alginate capsules are formed as indicated above. Semipermeable membranes are deposited in the surface layer of the capsules by suspending them in a 0.005% (w / v) aqueous solution of poly L lysine, (molecular weight 43,000 daltons) for 3 minutes.

The resulting capsules are suspended in CMLR-1969, supplemented with 10% fetal calf serum. The previous steps are all performed at 37 ° C. after incubation via the same temperature, it is found that the capsules, if examined under a microscope, contain fibroblasts, which have undergone mitosis and show three-dimensional fibroblastic morphology within the microcapsules.

Selective rupture of the membranes EXAMPLE 5 Microcapsules from any of Examples 1-4 can be treated as follows to selectively disintegrate the capsule membranes without damaging the encapsulated core material. 10 ml portions of microcapsule suspensions, containing about 500-5000 capsules per ml, are allowed to settle and the suspension medium is removed by aspiration. The capsules are washed twice with Selt Solution. The washed capsules are then mixed with a 3.0 ml aliquot of saline containing heparin in varying concentrations as will be given below and 1.1% w / v CaCl 2. Capsules which have alginate enclosed therein, after this step is completed, exhibit a solid, shape-retaining inner core. The suspension is stirred at 37 ° C for 10 minutes, after which the capsules are allowed to settle, the supernatant layer is removed by aspiration and the capsules are washed twice with 3.0 ml of 0.15 ml. M NaCl. After aspiration of the second washing solution, the capsules are mixed with 2.0 ml of a mixed solution containing equal volumes of 10 ml of sodium citrate and 0.15 M NaCl pH = 7.4).

Capsule membranes which had been treated with 1000 units / ml heparin and vortexed in NaCl-Na citrate solution for 1 minute were completely disintegrated. The same result is achieved with capsules treated with 2000 units of heparin for 2 minutes, followed by 15-30 s of hand vortex. Lower concentrations of heparin are preferred as the possibility of cell damage is reduced. After dissolving the membranes, each membrane debris can be removed by aspiration and washing. After the liberated cells have been resuspended in the culture medium, they can be tested by the exclusion technique using the dye tryptan blue and will be found to be in a state of good health and viability, with only relatively few cells exhibiting color uptake.

EXAMPLE 6 Capsules prepared according to Example 3 are treated, after washing, with a 3.0 ml solution containing 1000 units / ml heparin and 1.0% AlCl 3 for 6 minutes with stirring. After aspiration of the overlying layer, the core material is released by swirling the capsules with a 0.1 M solution of sodium citrate for 30-90 s.

EXAMPLE 7 The procedure of Example 6 is repeated except that 0.1M EDTA (sodium form) at a pH of 7.0 is used instead of the sodium citrate, which gives a rapid decomposition of the capsule membranes.

EXAMPLE 8 Capsules prepared according to Example 3 are treated, after washing, with a 3.0 ml aqueous solution containing 10 mg / ml of polyvinyl sulphate (molecular weight about 50,000 daltons) and 1% CaCl 2. Post-treatment with 0.10 M sodium citrate gives a substantially complete dissolution of the capsules.

Other embodiments are within the scope of the following claims. 9.5

Claims (14)

10 15 20 25 30 35 454 181 19 PATENT CLAIMS A method of disintegrating a permeable capsule membrane, to release an encapsulated material, the capsule membrane comprising a matrix of a polycationic polymer and a polyanionic polymer, which polymers are bonded to each other by salt bridges between the anionic and cationic groups; characterized in that A. the membrane is exposed to a solution of polyvalent cations and a polyanionic stripping polymer having a sufficient charge to separate the salt bridges; B. that the cations are allowed to compete with the polycationic polymer for anionic sites on the polyanionic polymer, and that the polyanionic stripping polymer is allowed to compete with the polyanionic polymer for cationic sites on the polycationic polymer and C. that cations associated with the polyanionic reindeer; the polymer is separated after step B. 2. A method according to claim 1, characterized in that the separating step (C) is performed by exposing the membranes to a solution containing a chelating agent. 3. A method according to claim 2, characterized in that the chelating agent is selected from citrate ions and EDTA ions. 4. A method according to any one of claims 1-3, characterized in that the polycationic polymer comprises a plurality of amino groups. 5. A method according to claim 4, characterized in that the polyanionic polymer comprises an acidic, water-soluble rubber. 6. A method according to claim 5, characterized in that the polycationic polymer comprises an alginate. 10 15 20 25 30 35 454 181 20 _ e 7. A method according to claim 4, characterized in that the stripping polymer comprises heparin and that the cations in solution comprise Ca ++. 8. A method according to any one of the preceding claims, characterized in that the polycationic polymer is selected from: a) proteins having a plurality of amino acid units having free amino groups; b) proteins having a plurality of amino acid units * having free imino groups; c) polypeptides having a plurality of amino acid units having free amino groups; d) polypeptides having a plurality of amino acid units having imino groups; 'e) polyvinylamines; f) polyethyleneimines; g) polyethylene amines; and h) mixtures thereof. 9. A method according to claim 1, characterized in that the stripping polymer is selected from: a) polysulfonic acids; b) polyphosphoric acids; c) salts thereof; and d) mixtures thereof. 10. A method according to any one of the preceding claims, characterized in that the stripping polymer is a polymer having polysulfonic acid salt groups. 11. ll. A method according to any one of the preceding claims, characterized in that a polycationic stripping polymer having a higher charge density than the charge density of the polyanionic polymer of the capsule membrane is used. 12. A method according to claim 1, characterized in that the capsule membrane defines a microcapsule containing one or more living cells. 13. The method of claim 12, wherein the polycationic polymer comprises a protein, the water-soluble rubber comprises sodium alginate, the cations in solution comprise calcium and the polyanionic stripping polymer comprises heparin. 14. The method of claim 13, wherein the separating step is performed with citrate dissolved in a solution that is physiologically compatible with the cell or cells.