Encapsulation of chemically active biologically active materials and viable tissue

(54) Description Procedure for encapsulation of a core material within a selective permeable membrane and capsule (56) Published Publications: SE 426 245 (C07C 7/00)

Other Publications: Biomedical Applications of Immobilized Enzymes and Proteins, Vol.l (1977), 69-82.

(57) Summary:

Viable tissue, such as Langerhans mammalian or liver tissue islets, encapsulated within a selectively permeable membrane, allowing passage of oxygen, amino acids and nutrients required for tissue preservation and ongoing metabolism, but impermeable to bacteria, lymphocyte and proteins. Above a selected level to exclude potentially harmful large molecules, such as immunoglobulins »With the method, an insulin-producing system can be produced, where mammalian cell islets are preserved in a viable and protected state within the capsule and hormones are secreted. Even mammalian liver tissue can be encapsulated for use as a body fluid detoxification system. The encapsulated liver tissue is shielded from plasma proteins of the type responsible for immunological rejection and from leukocytes and bacteria, but toxins and nutrients required for preservation and continued normal metabolism of the tissue pass freely through the membrane. The capsules may be injected into a suitable site in a mammalian body and act as an artificial organ, for example an artificial pancreas.

The method comprises suspending the material to be encapsulated in a physiologically compatible medium, such as a culture medium, which contains a water-soluble substance which can be gelled into droplets to form stand-alone, preservative temporary capsules, formation of a selectively permeable membrane around the temporary capsules, and optional rewetting of the gelled pre-fed food. The process can also be used for encapsulation of chemically active substances, such as activated carbon particles and labile biological materials.

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Background of the invention

The present invention relates to a method of encapsulating tissue or individual cells so that they remain viable and in a protected state within a membrane, which is permeable to nutrients, ions, oxygen and other materials needed to both preserve the tissue and maintain its tissue. normal metabolic functions, but impermeable to bacteria, lymphocytes and large proteins of the type responsible for immunochemical reactions resulting in rejection. The invention further relates to the capsules thus obtained.

The method of the invention enables production of, for example, an insulin-producing system or other hormone-producing system, since it allows encapsulation of mammalian pancreatic beta cells, alpha cells, intact Langerhans cell islands, and other tissues or tissue fractions that secrete hormones. The capsules can be suspended in a culture medium and they secrete hormones for an extended period of time. The capsules can also be used as an artificial pancreas, which can be implanted, for example, by injection, into a diabetic mammal, and it functions in vivo to secrete insulin and other hormones in response to ambient sugar concentration.

It is believed that the technology lacks methods for encapsulating · living tissue so that the tissue remains viable. Attempts to achieve this are hindered by the conditions required for capsule membrane formation, which are typically hostile to living systems. A technique is described for encapsulating labile biological materials within a semi-permeable membrane, which technique is capable, for example, of encapsulating enzymes within a membrane from which the enzyme cannot disappear while allowing free passage of the substrate of the enzyme. Although the technique involves reaction conditions

448 060, which preserves the delicate functional ability of biological materials, however, there is no suggestion that living tissue can be encapsulated.

Encapsulated living cells, organelles or tissues have many potential uses. For example, the live material encapsulated within a selectively permeable membrane can be preserved in a permanently sterile environment and can be protected from direct contact with large, potentially destructive molecular species, while allowing free passage of low molecular IQ tissue nutrients and metabolic products.

Thus, the development of such encapsulation technique could lead to systems for generating useful hormones such as insulin. In such systems, the mammalian tissue responsible for the production of the material would be encapsulated in such a way as to allow free passage of nutrients and metabolic products through the membrane while preventing passage of bacteria. If membrane permeability could be regulated, it is possible that this attack on the problem could also lead to artificial organs that could be implanted in a mammalian body, e.g., a diabetic, without rejection and with controlled hormone release, e.g., insulin release, controlled by glucose concentration.

Various attempts have been made to produce artificial organs suitable for implantation in mammalian bodies by providing a mechanical, selectively permeable barrier, such as a Millipore diffusion chamber or capillary tube chamber, around tissue excised from a donor. Such artificial organs usually require surgical implantation. In addition, the mammalian body's protective mechanisms isolate the implant, typically by clogging pores by overgrowth of fibroblasts.

Summary of the Invention

The present invention provides a method of encapsulating core materials, such as living tissue, individual cells or biologically active materials, in a semipermeable membrane. The basic principle involves suspending the tissue to be encapsulated in a physiologically compatible medium, ττί

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448 060 which contains a water-soluble substance which can be rendered insoluble in water, i.e., gelated, to provide a temporary protective environment for the tissue. The medium is then formed into small droplets containing the tissue and gelled, for example, by changing the conditions of temperature, pH or ionic environment, so that the temporary capsules formed are then subjected to a treatment which can be a known treatment which results in the production of membranes with a controlled permeability (including impermeability) around the mold retaining temporary capsules.

The invention thus relates to a method for encapsulating a core material within a selectively permeable membrane without significantly damaging the core material, which method comprises

Ά. adding the core material to a solution of a water-soluble substance which can be gelled to form a coherent, mold retaining mass;

B. forming the solution into drops;

C. the droplets are gelled to form free-standing, mold-preserving masses; and

D. that a selectively permeable membrane is formed around the mold retaining masses by cross-linking or polyaddition of the surface layers of the mold retaining masses.

The invention further relates to a capsule comprising a selectively permeable membrane defining an inner capsule space containing one or more viable, healthy living cells capable of undergoing metabolism, the membrane comprising a polysaccharide having a plurality of anionic groups which are salt bound to a polymer having a plurality of cationic groups.

The temporary capsules can be made from any non-toxic, water-soluble substance which can be gelled to form a mold retaining mass by changing the conditions of the medium in which it is placed, and also comprises a plurality of groups which are readily ionized to form anionic or cationic groups. The presence

448 060 of such groups in the polymer allows the surface layer of the capsule to be crosslinked to provide a permanent membrane when exposed to polymers containing multiple functionalities of the opposite charge.

The currently preferred material for forming the temporary capsules are polysaccharide gums, either natural or synthetic, of the type which can be a) gelled to form a mold retaining mass by being subjected to a change in conditions, such as a pH change, or by being exposed to multivalent cations such as Ca ++; and b) permanently crosslinked or cured by polymers containing reactive groups, such as amine or imine groups, which can react with acidic polysaccharide constituents. The currently preferred rubber is alkali metal alginate. Other water-soluble gums which may be used include guar gum, gum arabic, carrageenan, pectin, tragacanth gum, xanthan gum or acid fractions thereof. When encapsulating thermally hard-working materials, gelatin or agar can be used instead of rubber.

The preferred method of forming the droplets is to force the rubber-nutrientia tissue suspension through a vibrating capillary tube located within the center of the vortex created by the rapid stirring of a solution of a multivalent cation. Droplets ejected from the capillary tip immediately come into contact with the solution and gels as spheroidal shaped bodies.

The preferred method of forming a permanently selective permeable membrane around the temporary capsules is to cross-link, surface layers of a gelled rubber of the type having free acid groups with polymers containing acid-reactive groups, such as amine or imine groups. This is typically done in a dilute solution of the selected polymer. In general, the lower the weight of the polymer, the greater the penetration into the surface of the temporary capsule and the greater the penetration, the less permeable the resulting membrane. Permanent cross-links are generated as a consequence of salt formation between cross-links?

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<td></td><td>5 acid-reactive groups of the polymer and polysaccharide gum</td>

<td></td><td>acid groups. Within certain limits, semi-permeability can be regulated by adjusting the molecular weight of the crosslinking polymer, its concentration and the reaction time. The cross-linking</td>

<td> 5</td><td>polymers that have been used successfully include polyethyleneimine. and polylysine. The molecular weight may vary, depending on the degree of permeability required, between about 3,000 and 100,000 or more. Good results have been obtained using polymers having an average molecular weight.</td>

<td> 10</td><td>cooling weight in the order of 35,000. The capsules may be arranged to have a selected in vivo useful life by careful selection of the crosslinking polymer. Proteins or polypeptide bridges, such as polylysine, are readily attacked in vivo, leading to</td>

<td> 15</td><td>tive rapid destruction of the membrane. Bridgers that are not readily digested in mammalian bodies, such as polyethyleneimine, Lead to membranes that last longer. By selecting the crosslinking polymer or by crosslinking simultaneously or in succession with two or more such materials,</td>

<td> 20</td><td>possible to preselect the period of time that the implanted tissue remains protected. With certain materials used to form the temporary capsules, it is optionally possible to improve mass transfer within the capsule after the formation of the individual capsules.</td>

<td> 25</td><td>the human membrane by resetting the conditions under which the material is in liquid form, for example, removal of the multivalent cation. This can be done by ion exchange, such as immersion in phosphate buffer wire physiological saline or citrate buffer. In some situations, such as</td>

<td> 30</td><td>when it is desirable to preserve the encapsulated tissue or when the temporary gelled capsule is permeable,</td>

<td></td><td>it may be appropriate to leave the encapsulated rubber in the cross-linked, gelled state. An alternative method for the formation of moms includes</td>

<td> 35</td><td>an interface polycondensation or polyaddition similar to the process described in U.S. Patent Application Serial No. 606,166. This approach involves making one</td>

448 Suspension of temporary capsules in an aqueous solution of the water-soluble reaction component of a pair of complementary monomers capable of forming a polymer. Subsequently, the aqueous phase is suspended in a hydrophobic liquid in which the complementary reaction component is soluble. When the second reaction component is added to the two-phase system, polymerization occurs at the interface. The permeability can be controlled by controlling the addition of the hydrophobic solvent and the concentration of the reaction components. Yet another way to form a selectively permeable membrane is to include an amount of protein in the temporary capsule, which can then be crosslinked in the surface layers by exposure to a solution of a crosslinking agent such as glutaraldehyde.

The foregoing method has been used to encapsulate viable Langerhans cell islands which, in a medium containing nutrients and other materials necessary to maintain viability and support in vitro metabolism 'of the tissue, secrete insulin in the presence of glucose. Encapsulated tissue has been maintained in a viable condition for 3 months. Also, liver cells have been encapsulated and have been shown to be in a physiologically active state. The present invention also provides a tissue implantation method that does not require surgical intervention and overcomes many of the immune rejection problems. In accordance with the invention, the capsules are injected into a suitable site in a mammalian body and function normally until the tissue dies or until natural body processes succeed in isolating the capsules so that substances required for the tissue to live are no longer available. At this stage, fresh tissue can be readily provided by another injection, as surgical intervention is not required for implantation. Accordingly, the mammalian body can be provided with the specialized function of the tissue for as long as desired.

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In a preferred embodiment of the invention, mammalian Langerhans cell islands or cell preparations containing selected amounts of alpha, beta and / or delta cells from cell islands are encapsulated in polylysine and polyethylene imine crosslinked alginate membranes. These can be periodically injected, for example, into the peritoneal cavity of a diabetic mammalian body and function as an artificial pancreas.

Accordingly, a primary object of the invention is to provide a method for encapsulating living cells, organelles or tissue in a membrane permeable to nutrients and other substances required for maintenance and metabolism as well as for metabolic products but impermeable to bacteria and for substances having a molecular weight above a selected level, so as to exclude agents responsible for immunological rejection of the foreign tissue. Other objects of the invention include providing encapsulated living tissue useful for generating hormones, such as insulin, and for carrying out complex chemical changes characteristic of the in vivo tissue, for providing an insulin-producing system for providing a body fluid detoxification system. and for providing encapsulated activated carbon.

Applications of the invention are to provide a method for implanting living tissue into mammalian bodies and a non-surgical tissue implantation technique. Yet another application of the invention is to provide a method of encapsulating living tissue which allows the manufacture of capsules having a high surface area to volume ratio and membranes with a predetermined in vivo residence time. Another application of the invention is to provide an artificial pancreas.

These and other applications and features of the invention will become apparent from the following description of certain preferred embodiments and. from the drawing.

Brief description of the drawing

The drawing shows schematically a preferred method for

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live tissue encapsulation suitable for use in the method of the invention and the microcapsule product.

Description of preferred embodiment

The tissue, organelle or cell to be encapsulated is prepared according to prior art techniques in finely divided form and suspended in an aqueous medium suitable for maintaining and supporting the ongoing metabolic processes of the particular tissue in question. Suitable media for this purpose are commercially available. The average diameter of the material to be encapsulated can vary widely from less than 1 μπι to several mm. Mammalian Langerhans' cell islands typically have 140 - 200 µm diameter. Of course, individual cells, such as pancreatic beta cells, alpha cells, delta cells or various ratios thereof, the entire Langerhans cell, individual hepatocytes, organelles or other tissue units can be encapsulated as needed. Microorganisms can also be encapsulated, as well as non-living materials and biological materials.

Continuous viability of such living material depends, inter alia, on the availability of required nutrients, oxygen transfer, the absence of toxic substances in the medium and the pH of the medium. To date, it has not been possible to maintain such living material in a physiologically compatible environment while simultaneous encapsulation has been performed. The problem has been that the conditions required for membrane preparation have been lethal or deleterious to the tissue, and no membrane formation method has been expected where the tissue can survive in a healthy state. It has now been discovered that some water-soluble substances, which are physiologically compatible with living tissue and can be rendered water insoluble to form a form-retaining, coherent mass, can be used to form a temporary capsule or a protected barrier layer around tissue particles. Such a material is typically added to the tissue culture medium, typically at low concentration. The solution is then formed into droplets containing the tissue together with its holding medium and then immediately rendered water insoluble and gelled, at least in a surface layer. Thereafter, the mold retaining temporary capsule is given a permanently selective permeable membrane. If the material used to form the temporary canister permits, the canister contents can be returned to liquid after the formation of the permanent membrane. This is done by restoring the conditions in the medium at which the material is soluble.

The material used to form the temporary capsules may be any non-toxic, water-soluble material which, upon changing the ambient temperature, pH or ionic environment or concentration, can be transferred to a mold retaining mass. Preferably, the material also contains a plurality of slightly ionized groups, for example carboxyl or amino groups, which can be rea. by salt formation with polymers containing a variety of groups which ionize to form groups of opposite charge. As will be explained below, this type of material enables the deposition of one. permeable membrane with a selected porosity and a selected in vivo life of the surface layer of the temporary capsule.

. The presently preferred materials for forming the temporary capsules are water-soluble, natural or synthetic polysaccharide gums. Many such materials are commercially available. Typically, they are extracted from vegetables and are often used as additives to various foods. Sodium alginate is the currently preferred water-soluble rubber. Other useful gums include guar gum, gum arabic, carrageenan, pectin, tragacanth, xanthan gum or their acidic fractions.

These materials include glycoside-linked saccharide chains. Many contain free acid groups, which are often present in alkali metal ion form, for example sodium form. If the alkali metal ion is replaced by a multivalent ion such as calcium or strontium, the liquid, water-soluble polysaccharide molecules are crosslinked to form a water-insoluble, form-preserving gel which can be resolubilized by removal of the ions by ion exchange or via one. sequestrants. Although substantially any multivalent ion capable of forming a salt is useful, it is preferred to use physiologically compatible ions, for example calcium. This = tends to preserve the tissue in a living state. Other multivalent cations can be used for less fragile materials.

Other rubbers can be switched between the water-soluble and gelled, water-insoluble state simply by changing the pH of the medium in which they are dissolved.

A typical tissue-tissue medium-rubber solution composition comprises equal volumes of tissue in its medium and a 1-2% solution of the rubber in physiological saline solution. When sodium alginate is used, a 1.0 - 1.5% solution has been used successfully.

When encapsulating materials that can withstand changes in temperature, gelatin or agar can be used to form the temporary capsules. These can be gelled by injection into a low temperature environment. Other water-soluble substances, such as hydroxyethyl methacrylate, may also be used.

In the next step of the encapsulation process, the rubber solution containing the tissue is formed into drops of a desired size. Thereafter, the droplets are gelled immediately to form spherical or spheroidal masses. Apparatus for performing these latter steps is shown at step BC of the drawing. A beaker 10 containing an aqueous solution of multivalent cation, e.g., 1.5% CaCl 3 solution, is equipped with a magnetic stir bar 11 and stirrer 12. The stirring mechanism is actuated to provide a vortex 14 with hollow center 16. A capillary tube 18 with a selected inner diameter is placed within the hollow region 16 of the vortex and equipped with a vibrator 20. The suspension, which contains tissue and the solubilized rubber, is fed through the capillary. The surface tension effect, which would induce the formation of relatively large droplets, is minimized by the vibrator * so that the droplets, shown at 22, are of a size

448 060 ii comparable to the inner diameter of the capillary, is shaken off the capillary tip. These come into contact with the solution immediately, absorbing calcium ions. This results in cross-linking of the gel and in the formation of a form-preserving, high viscosity-protecting temporary capsule containing the suspended tissue and its medium. The capsules are collected in the solution as a separate phase and separated by aspiration.

In an alternative embodiment of the process, a small amount of polymer of the type used to permanently cross-link the rubber in the solution is incorporated together with the multivalent ions (or other solution capable of gelling the particular rubber used). This results in the formation of permanent cross-links.

Capsules of this type .har. Some benefits if the goal is to preserve the tissue.

In the next step of the process, a selective permeable membrane is deposited around the surface of the temporary capsules. There are a variety of methods available for carrying out this step, some of which are known before. For example, interface polymerization techniques can be utilized. In interface polymerization, a pair of at least difunctional mutually reactive monomers, or a monomer and a relatively low molecular polymer, one of which is soluble in polar solvents such as water and the other soluble in hydrophobic solvents such as hexane, are reacted at the interface. of a water-in-oil emulsion. In accordance with the process described in U.S. Patent Specification No. 606,166, the material to be encapsulated in water is suspended or dissolved together with the water-soluble component of the reaction, the aqueous phase is emulsified in a hydrophobic solvent and the complementary monomer is added to the continuous system. phase so that polymerization occurs around the aqueous droplets. By controlling the type of solvent in the continuous phase and the concentration of the reaction component contained therein, it is possible to exert control.

448 060 of pore size and producing selectively permeable microcapsules. .

This technique can be used in accordance with the present invention if the water-soluble reaction component is dissolved in an aqueous solution and the solution is used to suspend the temporary capsules. This liquid suspension is then emulsified in, for example, hexane or in a hexane-chloroform mixture. Then, the complementary monomer is added, preferably in increasing amounts, to induce interface polymerization at the surface of the aqueous droplets. Due to the gelled mass of polysaccharide surrounding the suspended tissue, and especially if suitably buffered polyfunctional amino group-containing polymers, such as certain proteins, are used as the water-soluble reaction component, the process is such that the tissue survives the encapsulation in a fresh state. Substances useful in forming the membranes with the polyfunctional amines include diacids, diacid halides and multifunctional sulfonyl halides. In addition to the polyamines, diamines, polyols and diols can be used. Molecules containing a plurality of amine groups may also be crosslinked with glutaraldehyde to form a membrane. Another useful membrane formation method is interface polymerization which utilizes polyaddition reactions. In this case, for example, multifunctional amines absorbed in the surface layers of the temporary capsules are reacted with epichlorohydrin, epoxidized polyesters or diisocyanates.

The preferred method of forming the membrane, shown as step D of the drawing, is to permanently crosslink the surface layers of the droplets by subjecting them to an aqueous solution of a polymer containing groups reactive with functionalities in the gel molecules. Some long-chain quaternary ammonium salts can be used for this purpose in certain circumstances. When acid gums are used, polymers containing acid-reactive groups such as polyethyleneimine and polylysine can be used. In this situation, the polysaccharides are cross-linked by interaction between the carboxylic groups and the amine groups. The permeability can advantageously be controlled by selecting the molecular weight of the crosslinking polymer used. For example, a solution of low molecular weight polymer, for a given period of time, penetrates further into the temporary capsules than does a high molecular weight polymer. The degree of penetration of the cross-linking polymer is correlated to the resulting permeability. In general, the higher the molecular weight and the smaller the penetration, the greater the pore size. Generally, polymers having molecular weights in the range of 3,000 to 100,000 daltons or greater may be used, depending on the duration of the reaction, the concentration of the polymer solution and the degree of desired permeability. In a successful set of reaction conditions, using average molecular weight polylysine of about 35,000 daltons, reaction for 2 minutes, with stirring, involved a physiological saline solution containing 0.0167% polylysine. Optimal reaction conditions suitable for controlling permeability in a given system can be readily inhibited: empirically inhibited without inventive work.

The choice of crosslinking agent (s) also determines the in vivo residence time of the capsules. In the system described above, the permanent capsule membrane comprises polysaccharide (an easily digestible substance) crosslinked with one or both of a polypeptide or a protein, eg polylysine, or a synthetic substance, eg polyethyleneimine. Polymers vary according to the rate at which they can be dispersed in vi^iro. Some are digested without difficulty, eg protein; others slowly decompose and still others remain in disrepute. The method of the invention envisages cross-linking with one or more polymers to produce capsules at a selected dissolution rate in vivo, which generally range from a few to a few hours or days to substantial resistance. The example that follows describes how to make capsules that remain intact for at least about 2 months within the peritoneal cavity of rats. However, the invention is not limited to these features.

448 060 different capsule membranes and also to capsules with this degree of in vivo life. In fact, the optimal in vivo life of the microcapsules depends on their intended use and their site of implantation. Those skilled in the art will be able to empirically prepare microcapsules with a selected in vivo lifetime without inventive work, on the basis of this disclosure.

At this stage of the encapsulation, capsules may be collected which comprise a permanently selective permeable membrane around a gelled solution of rubber, tissue compatible culture medium, and tissue particles. If the intention is to preserve the tissue only in a protective environment, no further steps need be performed. However, if mass transfer is to be promoted within the capsules and through the membranes, it is preferred to return the gel to liquid to its water-soluble form. This can be done by restoring the conditions under which the rubber is a liquid, such as changing the pH of the medium or removing the calcium or other multifunctional cations used. In the gels, which are insoluble in the presence of the multivalent cations, the medium in the capsule can be resolubilized simply by immersing the capsules in phosphate buffered physiological saline solution containing alkali metal ions and hydrogen ions. Monovalent ions are exchanged for the calcium or other multi25 functional ions within the rubber when, as shown in step E of the drawing, the capsules are immersed in the solution with stirring. Other salts, such as sodium citrate, can be used for the same purpose.

Finally, depending on the type of technique used to form selectively permeable membrane, it may be desirable to treat the capsules to bind free amino groups of the type that would otherwise give the capsules a tendency to clump. This can be done, for example, by immersing the capsules in a solution of sodium alginate.

The invention permits injection of encapsulated, finely divided tissue, multicellular fractions thereof, or individual cells, into a suitable site within a mammalian body.

448 060 for the purpose of providing the body, at least temporarily, with the specialized physiological function of the tissue. The method has the dual advantages of eliminating the need for surgical implantation (although the capsules can be implanted surgically if desired) and of successful management of the problems of immune rejection and natural physical isolation. Preferably, the capsule membranes consist of substances which are digested after the tissue has died out. As stated above, this can be accomplished by utilizing a bridge former that resists in vivo degradation to achieve a given useful in vivo life.

From, the foregoing, it will be apparent that the encapsulation method of the invention and consequent implantation techniques can be practiced using a variety of reagents and encapsulated materials and can be significantly varied without departing from the scope and idea of ​​the invention. Accordingly, the following examples should in all respects be considered as illustrative and not as limiting.

Example 1

Langerhans' cell islets were obtained from rat pancreas and added to a complete tissue culture (CMRL-1969 Connaugt Laboratories, Toronto, Canada) at a concentration of about 10³ cell islands per ml. The tissue culture contained all the nutrients required for continued viability of the cell islets as well as the amino acids utilized by the beta cells to produce insulin. Four tenths of a milliliter of the cellulose suspension was then added to half a milliliter of 1.2% sodium alginate (Sigma Chemical Company) in physiological saline.

Then, 80 ml of a 1.5% calcium chloride solution was placed in a 150 ml beaker on a stirrer and stirred at a rate which induced the formation of a cone-shaped vortex at its center. A glass capillary with a gradually decreasing diameter, terminating at a tip with an inner diameter of about 300 µm, was then equipped with a vibrator (60 periods per second). The capillary tip was then placed within the center of the vortex, the vibrator turned on

448 060 and the sodium alginate culture medium tissue suspension were thereby forced by an infusion pump. Drops in the order of 300 - 400 µm in diameter were discharged from the tip of the capillary and immediately entered the calcium solution.

After 10 minutes, the stirrer was switched off and the supernatant solution was removed by aspiration. The gelled capsules were then transferred to a beaker containing 15 ml * of a solution comprising one part of a 2% 2- (cyclohexylaminojetansulfonic acid solution in 0.6% NaCl (isotonic, pH = 8.2) diluted with 20 parts). 1% CaCl₂· After 3 minutes of immersion, the capsules were washed twice in 1% CaCl₂·

The capsules were then transferred to a 32 ml solution containing 1/80 of 1% polylysine (average molecular weight 35,000 AMU) in physiological saline. After 3 minutes, the polylysine solution was decanted. The capsules were then washed with% CaCl₂ and then suspended for 3 minutes in a solution of polyethyleneimine (molecular weight 40,000 - 60,000) prepared by diluting a 3.3% polyethyleneimine stock solution in morpholine propane sulfonic acid buffer (0.2 M, pH = 6) with sufficient 1% CaCl₂ to give a final polymer concentration of 0.12%. The resulting capsules, with permanent selective membranes, were then washed twice with 1% CaCl₂, 2 times with physiological saline and mixed with 10 ml of a 0.12% alginic acid solution.

The capsules resisted clumping and many were seen to contain

Langerhans' cell islands. Gel in the interior of the capsules was returned to liquid by immersing the capsules in a mixture of physiological saline and citrate buffer (pH - 7.4) for 5 minutes. Finally, the capsules were suspended in CMLR-69 medium.

Under a microscope, these capsules had an appearance, as shown in the drawing. They included a very thin membrane 24 enclosing a cell island 26 within which individual cells 28 can be seen. Molecules with a molecular weight of up to about 100,000 can pass through membrane 24. This allows oxygen, amino acids, nutrients and plasma components used in the culture medium (e.g., fetal calf plasma components) to reach the islets and allows the secretion of insulin.

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Example 2

After repeated washings in physiological saline, the microcapsules prepared in accordance with Example 1, which contained about 15 cell islets, were suspended in 3 ml.

CMRL in 1969. When the capsules were 8 days old, in the presence of 600 mg / dl glucose, at one run, they secreted 67 units / ml of insulin for 1.5 hours. In a second run, 68 units / ml of insulin were generated at the same time. One week old capsules, in the same medium, but in the presence of 100 mg / dl glucose, secreted 25 units / ml insulin in 1.2 hours in the first run, and the secretion in the second run 10 units / ml.

Example 3

Diabetic rats with blood glucose levels in the area

500 700 mg / dl were each treated with about 10 cell islets encapsulated as set forth in Example 1 and suspended in physiological saline. The capsules were inserted by injection into the peritoneal cavity, using a No. 19 needle attached to a syringe. Blood sugar levels were analyzed daily and found to be uniformly below 300 mg / dl. The animals killed after 2 months showed no signs of toxic reaction around the implant site. After 2 months of in vivo life, the capsules removed from the sacrificed animals were intact and showed no evidence of degradation.

Example 4

Encapsulation of hepatocytes

The procedure of Example 1 was repeated except that 0.5 ml of a liver cell suspension in Hank's solution was used instead of the 0.4 ml cell suspension. The ongoing viability of the liver tissue had been demonstrated by the color exclusion technique (trypan blue exclusion).

It is known that liver tissue, in vitro, can ingest toxins from its environment. Toxins with a sufficiently low molecular weight to pass through the semipermeable membranes are ingested and therefore destroyed by the tissue. Virtually all of the toxins treated in the liver are low molecular weight materials. However, the toxins may be protein-complex bound. Capsule permeability can be varied as needed.

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Example 5

The procedure of Example 1 was repeated except that particulate activated carbon was suspended directly in the sodium alginate solution, omitted 1/2 ml of tissue suspension and that polylysine with an average molecular weight of 35,000 was used as a brewing agent. As long as the carbon particles are smaller than the minimum capillary inner diameter of the droplets used, the large surface area carbon is surrounded by a selectively permeable membrane. These effectively prevent the escape of carbon pieces or dust and can nonetheless be used to absorb materials of medium molecular weight (up to about 2,000 daltons) from fluids passing around the capsules.

The utility of the method has been demonstrated with other living cells, including red blood cells, using serum as a medium, sperm cells, using semen as the medium and bakery yeast. Those skilled in the art will recognize that a variety of materials may be encapsulated other than those specifically disclosed herein, and that permeability may be regulated as needed for selected applications of the process. Accordingly, other embodiments are within the scope of the claims.

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