WO1988000237A1

WO1988000237A1 - Covalent membranes

Info

Publication number: WO1988000237A1
Application number: PCT/US1987/001495
Authority: WO
Inventors: Franklin Lim; Lloyd Thomas Hall, Iii
Original assignee: Damon Biotech, Inc.
Priority date: 1986-06-27
Filing date: 1987-06-29
Publication date: 1988-01-14
Also published as: AU7646087A

Abstract

A process for encapsulating a gel bead containing core material within a permanent, covalently cross-linked semipermeable membrane. The process includes the steps of: activating a first compound possessing multiple functional groups by reaction of the first compound with a polyfunctional activating agent; placing a core material in a solution of a water-soluble substance which can be reversibly gelled, and which will covalently react with the activated first compound; forming a resulting solution into droplets: gelling the droplets to produce discrete shape retaining gel beads; and dropping the beads into a solution of the activated first compound to substantially instantaneously form a permanent covalent semipermeable membrane about the beads.

Description

COVALENT MEMBRANES BACKGROUND OF THE INVENTION

The present invention relates to microcapsules of semipermeable membrane, and more particularly to membranes which are formed covalently.

The production of microcapsules of semipermeable membrane which are useful in biological applications, such as for the icroencapsulation of cells, is known. For example, U.S. Patent No. 4,352,883 to Franklin Lim (incorporated herein by reference) discloses a microencapsulation technique which can be used to encapsulate solid or liquid material within semipermeable or substantially impermeable capsule membranes. The process disclosed in the '883 patent begins by suspending the core material in a solution of a water-soluble substance which can be reversibly gelled. The resulting solutio is formed into droplets which are gelled to produce discrete shape-retaining temporary capsules. A polyionically bonded semipermeable membrane is then formed around the temporary capsules, followed by reliquifying the gel within the capsules.

Core materials discussed in the '883 patent include living cells and finely divided living tissue which are suspended in an aqueous medium which is physiologically compatible with the cells or tissue. The preferred water-soluble substance therein for forming the temporary capsules is a gum, a preferred gum being sodium alginate. The temporary capsules are formed by subjecting the droplets to a solution of ultivalent cations, such as an aqueous solution of calcium chloride. In the preferred embodiment therein, the capsular membrane is formed by contacting the temporary capsules with a polymer of a molecular weigh between 3000 and 100,000 daltons which has free amino groups which react with the free carboxyl groups of the alginate. This results in reversible, non-covalent (polyioniσ) cross-linking of the surface layers of the temporary capsules. Preferred crosslinking polymers are polylysine and other cationic polyamino acids. In » the embodiment where sodium alginate is the water- soluble substance, the gelled core material is reliquified by removal of the calcium ions contained in the capsules, thereby resolubilizing their interior. Other patents relating to this same general system of forming membranes of the polyelectrolyte complex type and various modifications thereof are U.S. Patents 4,391,909; 4,407,957; 4,409,331; and 4,495,288 to Franklin Lim, all incorporated herein by reference. Although this technology allows production of acceptable microcapsules, a need has continued to exist for a process capable of producing microcapsules which include a true permanent polymeric membrane. In addition, a desire has also existed for a process which is capable of forming a bilaminar membrane.

SUMMARY OF THE INVENTION

The present invention is a process for encapsulating a gel bead containing core material within a permanent, covalently cross-linked semipermeable membrane. The process includes the steps of: activating a first compound possessing multiple functional groups by reaction of the first compound with a polyfunctional activating agent; placing a core material in a solution of a water- soluble substance which can be reversibly gelled, and which will covalently react with the activated first compound; forming a resulting solution into droplets; gelling the droplets to produce discrete shape retaining gel beads; and dropping the beads into a solution of the activated first compound to substantially instantaneously form a permanent covalent semipermeable membrane about the beads.

DETAILED DESCRIPTION OF THE INVENTION

The process of the present invention employs the polyelectrolyte complex capsules or gelled material of the prior art (for example, of the type described in U.S. Patent No. 4,352,883). The polyelectrolyte complex capsule or gelled material is modified at its outer surface by means of reaction with an activated polymer such as polyacrylic acid modified with

Woodward's Reagent K, polymeric dextran modified with sulfoSANPAH (sulfosuccinimidyl 6-(4'-azido-2'- nitrophenylamino) hexanoate) , or other appropriately chosen activated polymer. In the instance where a polyelectrolyte complex capsule is so treated, the result of the process is to produce a bila inar, i.e., two-layer, capsule membrane, the inner region of which is characterized by polyionic bonding, and the outer region of which is characterized by covalent bonding. The inner, polyionic region of this bilaminar structure may subsequently be disrupted as necessary by treatment with appropriately chosen polyvalent ions or low molecular weight ionic species. By low molecular weight is meant under about 1000 daltons. The outer membrane, due to its covalent character, will be substantially unaffected by this treatment and will therefore serve to maintain the overall structure of the capsule. The permeability of the resultant membrane may, however, differ substantially from the permeability of the original bilaminar system.

The relative thicknesses and permeabilities of the two regions of the bilaminar membrane may be controlled by altering the nature, concentrations and exposure times of the reacting species. It should be recognized, though, that the inner polyionic region acts as a reactant in the formation of the outer, covalent region of the bilayered membrane structure. For this reason, increases in the thickness of the covalent region may occur to some extent at the expense of the inner, polyionic layer.

In the instance where a gelled material is modified according to the process of the invention, a unilaminar covalent membrane surrounding the gelled material is produced. Two means of generating such a unilaminar membrane are described below. In the first case, the gelled material to be encapsulated (for example, chitosan acetate gelled with sodium citrate) is treated with an activated polymer solution (for example, polyacrylic acid derivatized with Woodward's Reagent K) under conditions that permit the formation of a covalently cross-linked membrane around the gelled core.

The required conditions are that the gelled material possess sites at which chemical reaction can occur under the available conditions of pH, temperature, and solvent composition, and further that the activated polymer possess moieties capable of covalently bonding to the reactive sites on the gelled material. The permeability of the resulting membrane varies according to the concentration of the reactants, the reaction time, and the abundance of reactive sites present on the reactive species.

In the second case the gelled material to be encapsulated is itself activated in such a manner as to allow its covalent reaction with an appropriately chosen polymeric reactant. As an example, beads of sodium alginate gelled in calcium chloride are derivatized with Woodward's Reagent K, and the resulting activated surface is treated with polylysine. The result of this process is the production of a gel bead surrounded by a covalently crosslinked polymer membrane.

The degree of crosslinking, and therefore the permeability of the resulting membrane, can be controlled by altering the concentrations and reaction times of the reactants, or by varying other experimental conditions in a manner appropriate to the reactive species in use. Examples of these other conditions are temperature, ionic composition of the medium and light intensity at the site of polymerization. In both the cases noted above, the gelled core of the product capsule may be reliquified if desired by the addition of appropriate ungelling agents. In the above examples, suitable ungelling agents would be calcium chloride and sodium citrate, respectively.

For example, microcapsules of the polyelectrolyte complex type can be prepared such that their core contains enzyme-antigen complexes having a unit molecular weight of about 5000 daltons. The polyelectrolyte complex membrane of such capsules would be prepared in such a way as to prevent the escape by diffusion of the core material. A covalent membrane is then placed around the polyelectrolyte complex membrane. The outer covalent membrane can be produced in such a way that it is permeable to the enzyme- antigen complexes contained in the core. To the outer covalent membranes can then be coupled antibody molecules which specifically bind the antigen portion of the enzyme-antigen complex of the core material.

In use, the capsules produced would be exposed to free specific antigen in aqueous medium, and binding of the antigen to the immobolized antibody would be allowed to occur. The microcapsules would then be removed and washed. The capsule would then be treated with a solution capable of disintegrating the inner polyelectrolyte complex membrane. Upon dissolution of the inner membrane the enzyme-antigen complexes of the core material are free to diffuse out of the capsule. The antigen moiety of such complexes can then bind to any unfilled binding sites of the immobilized antibody on the outer surface of the capsule. When this binding process is substantially complete the capsules are again washed leaving bound enzyme on the surface of the capsules and only washing solution in the core.

A chromogenic substrate can then be added to the system and the color change measured after the defined incubation period. The amount of color change can then be related to the amount of the free antigen originally added to the system. A test system based on this sort of approach but utilizing radiolabeled antigen rather than an enzyme-antigen complex could also be utilized. In either case, a simple, extremely sensitive, assay system results. With respect to the activated compound employed in the present invention, certain compounds can be used without modification, for example, polymeric aryl azides or n-hydroxy succinimide esters. In other cases, the compound may require activation or derivitization to produce reactive chemical groups capable of covalent bond formation with the functional groups of the other reacting compound or membrane surface. Typical examples of covalent bonds formed in this manner are ester bonds, peptide bonds, disulfide bonds, and bonds formed by free radical reactions.

Activation of functional groups may be accomplished by initiators or activators such as light or temperatures or by specific cross-linking reagents which functionally possess at least two types of reactive groups, hereafter referred to as "polyfunctional activating agents". Membrane pore size can be controlled by choosing or adjusting the density (number) and distribution of the reactive groups in the reactants, adjusting the concentration of the reactants, or adjusting the reaction conditions, such as time or temperature.

In one embodiment the process of the present invention produces microcapsules substantially instantaneously with a true covalently linked polymeric membrane, about a reversible polyelectrolyte complex type of membrane of the prior art. By "substantially instantaneously" is meant, in fewer than about five seconds. Preferably, production of the covalently linked polymeric membrane is complete in one second or less, particularly when living material is involved. Otherwise, the chemicals and conditions used may adversely affect its viability.

The tissues, organelles, or cells to be encapsulated in accordance with the process of the present invention are prepared in accordance with well- known prior art techniques. The material to be encapsulated is suspended in a solution of a water- soluble substance which can be reversibly gelled. Subsequent to encapsulation, a medium is utilized which is suitable for maintenance and for supporting the ongoing metabolic processes of the particular material involved. Media suitable for these purposes are available commercially. The average diameter of the material being encapsulated can vary widely between less than a micron to several millimeters. Thus, samples of materials as divergent in size as mammalian Islets of Langerhans (50-200 micrometers in diameter) , Herpes simplex virus (30-40 nanometers in diameter) , or onomeric immunoglobulins (molecular weight approximately 150,000 daltons) can all be encapsulated

SUBSTITUTESHEET by the process in this application. The core material to be encapsulated can be discrete living cells, viable tissue of plant or animal origin, or Protista such as viruses. For example, individual cells such as fibroblasts, leukocytes, pancreatic beta cells, alpha cells, delta cells, or various ratios thereof. Islets of Langerhans, individual hepatocytes, organelles, or other tissue units may be encapsulated as desired. Other cells which can be encapsulated include thymic cells, thyroid cells, liver cells (e.g., hepatocytes), adrenal cells, blood cells, lymphoid cells, and pituitary cells. Also, microorganisms may be encapsulated as well as non-living materials of biological or non-biological origin. The process of the present invention includes the in situ formation of a membrane on the surface of a capsule. This surface, if necessary, may be modified during the process, for example, by activation or by the addition of reactive sites. For example when one of the reactants is polyacrylic acid, the carboxylic acid moieties of the polyacrylic acid can be activated, for example, by reaction with Woodward's Reagent K (N-ethyl-5- phenylisoxazolium-3'-sulfonate) . While in this case the activating group does not form a part of the final product membrane, it is acceptable for the activating species to be incorporated into the final product membrane.

Woodward's Reagent K is a well known material for enzyme immobilization by the formation of water- soluble enzyme-polymer conjugates. Typical of such prior art enzyme immobilization conjugates are alpha- chymotrypsin on polyacrylic acid, carboxymethyl cellulose, or poly-L-glutamic acid. Although a preferred polyfunctional activating agent is Woodward's Reagent K, numerous other polyfunctional activating agents are known which will, for the purposes of the present invention, act in the same manner as Woodward's Reagent K, provided suitable reactants are chosen. Examples of other such polyfunctional activating agents include m-maleimidobenzoyl-N-hydroxysuccinimide ester, m-maleimidobenzoylsulfosuccinimide ester, N- succinimidyl-(4-iodoacetyl)aminobenzoate, succinimidyl 4-(N-maleimidomethyl)-cyσlohexane-1-carboxylate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1- carboxylate.

In the first step of one embodiment of the present invention, a hydroxyl group-containing polymer is activated by reaction with the photoactivated cross- linking agent sulfoSANPAH and then the amino groups of the temporary chitosan capsule surface react with the activated groups on the dextran polymer. Such photoactivation is typically accomplished over a period of 5 - 45 minutes. However, it must be emphasized that dextran polymers and chitosan acetate are only being used as examples of suitable polyfunctional reactants. The present invention is applicable generally to reactions between a wide variety of covalently reactive species, including, but not limited to, polyamino and polysulfhydryl compounds, polycarboxylic acids, polymeric aryl azides, and polymeric n- hydroxysuccinimide esters.

In the encapsulation process of the present invention the solution containing the core material is formed into droplets of a desired size. The drop formation may be conducted by known methods. An exemplary procedure follows.

A receiving vessel of a selected volume, which vessel contains one of the reactants, e.g., calcium chloride, is fitted with a stopper which holds a drop-forming apparatus. The apparatus consists of a housing having an upper air intake nozzle and an elongate hollow body friction fitted into the stopper. A 10 cc syringe equipped with a syringe pump is mounted atop the housing with, e.g., a 20G by 1-1/2" needle squared off at the end which passes through the length of the housing. The interior of the housing is designed such that the tip of the needle is subjected to a constant air flow which acts as an air knife. In use, with the syringe full of solution or suspension containing the material to be encapsulated, e.g., cells in a sodium alginate suspension, the syringe pump is actuated to incrementally force droplets of solution or suspension from the tip of the needle. Each drop is "cut off" by the air stream and falls approximately 2 to 10 cm into the vessel containing the gelling solution, e.g. aqueous calcium chloride. The distance between the tip of the needle and the surface of the solution in the vessel is great enough, in this instance, to allow the solution or suspension to assume the most physically favorable shape; i.e., a sphere

(maximum volume for minimum surface area) . Air within the vessel bleeds through an opening in the stopper. This results in the formation of a shape-retaining temporary capsule containing the tissue. The capsules collect in the solution as a separate phase and may be separated by decantation. The separated gel beads may then have a permanent covalent membrane formed directly around them or may first have a polyelectrolyte membrane formed, followed by the permanent covalent membrane.

Capsules formed by the process of the present invention have essentially the same utilities as those of the prior art, and thus also the same advantages, and are in fact useful in applications where material encapsulated solely by the polyelectrolyte complex type microcapsules of the prior art would not be useful because of potential adverse ionic interactions, or lack of permanency of the polyelectrolyte complex membrane.

The process of the present invention is useful for the encapsulation of biologically active material such as enzymes, hormones, and antibodies. The process of the present invention is also useful for the encapsulation of viable Islets of Langerhans. The resulting capsules, when placed in a medium containing the nutrients and other materials necessary to maintain viability and support in_Vitro metabolism of the tissue will maintain their complete physiological functional integrity. Other types of cells similarly can be encapsulated in a physiologically active state. The capsules of the present invention would also be useful for tissue implantation into a mammalian body. Another use for the present invention would be in the manufacture of an artificial or bioartificial organ. Similarly, the capsules of the present invention can be used in the culturing of anchorage dependent cells according to the method described in U.S. Patent 4,495,288, incorporated herein by reference.

Cell cultures encapsulated as described above may be suspended in culture media designed specifically to satisfy all of the requirements of the particular cell type involved and will continue to carry out normal metabolic processes in the capsules. Nutrients required by the cells will normally be of sufficiently low molecular weight so that their diffusion into the microcapsule will be substantially unimpeded by the membrane. However, the membrane may be constructed in such a way as to limit the escape of the cells' high molecular weight metabolic products, thereby allowing the isolation of these products from the relatively smaller volume of the intracapsular medium rather than the larger volume of the extracapsular medium. This concentrating effect of microcapsular membranes is an advantage that the present invention shares with the microcapsules of the prior art.

The encapsulated cells may be cultured under conditions of, e.g., temperature, pH, and ionic environment, identical to conventional cultures. Also, cell-produced products may be harvested from the extracapsular medium or from within the capsules by conventional techniques. Examples of cell produced products which may be harvested include insulin, glucagon, antibodies, prolactin, somatostatin, thyroxin, steroid hormones, pituitary hormones, interferons, FSH, and PTH.

In order to further illustrate the present invention and the advantages thereof, the following specific examples are given, it being understood that these examples are intended only to be illustrative without serving as a limitation on the scope of the present invention.

EXAMPLE 1 In 20 ml of saline is dissolved 0.2 g of T40 dextran (m.w. 40,000). To this is added 5 mg of SulfoSANPAH (sulfosuccinimidyl 6-(4'-azido-2'- nitrophenylamino)hexanoate) (a product sold by Pierce Chemical Company, P. 0. Box 117, Rockford, Illinois 61105) . The resulting solution is placed in a closed container with a high pressure mercury light source. The solution is irradiated for about 30 minutes while maintaining a temperature in the container of about

80°C. After cooling, the resulting activated dextran is rehydrated to 1% by volume of dextran by the addition of saline.

An approximately 100 microliter pellet of myeloma cells is suspended in two illiliters of one percent isotonic chitosan acetate in physiological saline (pH 5.8). The chitosan acetate is readily prepared by suspending 2 grams of chitosan in 200 ml of deionized water, adding 0.62 ml of acetic acid, mixing until the chitosan dissolves, and filtering to remove undissolved material.

The cell-containing chitosan acetate solution is added dropwise to a three percent by weight solution of sodium citrate (pH 6.8) to form polyionically bonded temporary capsules. The resulting temporary capsules are removed from the citrate solution, followed by the addition of an aliquot of activated dextran solution, and mixing for about 1 minute to produce a covalently bonded membrane around the polyionically bonded membrane. The cores of the capsuled are then ungelled by placing the capsules in a 0.65% by weight solution of calcium chloride (pH 6.2). However, the capsules remain intact, thereby demonstrating that the outer membrane is covalently bonded, since the interior ionically bonded layer disintegrates under these conditions.

EXAMPLE 2 A one mM solution of T40 dextran (m.w. 40,000) in saline is prepared. Approximately 5 mg of SANPAH (N-succinimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate) are suspended in approximately 20 ml of the dextran solution.

The resulting solution is irradiated for 15 minutes under a UV light source, and the resulting solid is redissolved in saline to approximately one percent by volume, and then the debris removed by centrifuging.

Gelled and ungelled microcapsules produced by the process of U.S. Patent 4,352,883, as described in "Microencapsulation of Living Cells and Tissues -

Theory and Practice", in Biomedical Applications of Microencapsulation, Franklin Lim, Ph.D, editor, CRC Press, Inc. Boca Raton, Florida, pages 139-141 (1984) , incorporated herein by reference, and containing hybridoma cells are suspended in the activated dextran supernatant for 1 minute at ambient temperature, and then washed twice in 1% saline.

Treated microcapsules which are agitated vigorously in 2,000 units per ml of heparin in 3% by weight citrate (pH 7.4) for 30 minutes reveal no change in appearance. Even shaking overnight at room temperature does not result in dissolution.

While the invention has been described in terms of various preferred embodiments, one skilled in the art will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof. Accordingly, it is intended that the scope of the present invention be limited solely by the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A process for encapsulating a gel bead containing core material within a permanent, covalentl cross-linked semipermeable membrane, the process comprising the steps of: A. activating a first compound possessing multiple functional groups by reaction of the first compound with a polyfunctional activating agent;

B. placing the core material in a solution of a water-soluble substance which can be reversibly gelled, and which will covalently react with the activated first compound;

C. forming the solution of step B) into droplets;

D. gelling the droplets to produce discret shape retaining gel beads; and

E. dropping the gel beads into a solution of the activated first compound to substantially instantaneously form a permanent, covalent semipermeable membrane about the gel beads.

2. The process of claim 1 wherein the first compound is a polycarboxylic acid.

3. The process of claim 1 wherein the activating agent is Woodward's Reagent K.

4. The process of claim 1 wherein the water- soluble substance is a gum.

5. The process of claim 4 wherein the gum comprises a polysaccharide containing free amino groups.

6. The process of claim 4 wherein the gum is chitosan acetate or hydrochloride.

7. The process of claim 2 wherein the polycarboxylic acid is a polyacrylic acid.

8. The process of claim 1 wherein the core material is composed of discrete living cells, viable tissues of plant or animal origin, or Protista.

9. The process of claim 1 wherein the core material is a biologically active material.

10. The process of claim 9 wherein the biologically active material is an enzyme, hormone, or antibody.

11. The process of claim 1 wherein the core material is a mammalian tissue selected from Islets of Langerhans, and individual cells thereof, suspended in a physiologically compatible tissue medium.

12. The process of claim 1 wherein the core material comprises living tissue in a physiologically compatible tissue medium.

13. A process for encapsulating a polyionically crosslinked microcapsule containing a core material within a permanent, covalently cross- linked semipermeable membrane, the process comprising the steps of: A. placing the core material in a solution of a water-soluble substance which can be reversibly gelled; B. forming the solution into droplets; C. gelling the droplets to produce discret shape-retaining gel beads;

D. forming polyionically bonded semipermeable membranes about the gel beads;

E. activating a reactant by reaction with polyfunctional activating agent; and

F. contacting the polyionically bonded semipermeable membrane surface with the reactant.

14. The process of claim 13 wherein the water-soluble substance is a gum.

15. The process of claim 14 wherein the gum comprises a polysaccharide containing free acid groups.

16. The process of claim 14 wherein the gum is an alkali metal alginate.

17. The process of claim 14 wherein the gum has free acid groups and the membrane formation step D is effected by contacting the gel beads with a polymer of a molecular weight between 3000 and 100,000 daltons and having free amino groups, the contacting being effective to form polyionic crosslinks between acid groups in a surface layer of the gel bead.

18. The process of claim 17 wherein the polymer used for crosslinking is polylysine, polyargenine, polyornithine, or polyethylenimine.

19. The process of claim 13 wherein the compound of step F is dextran of a molecular weight of between about 3,000 and about 100,000 daltons.

20. The process of claim 13 wherein the activating agent is sulfo-SANPAH or SANPAH.

21. The process of claim 13 wherein the core material is composed of discrete living cells, viable tissues of plant or animal origin, or Protista.

22. The process of claim 13 wherein the core material is a biologically active material.

23. The process of claim 22 wherein the biologically active material is an enzyme, hormone, or antibody.

24. The process of claim 13 wherein the core material is a mammalian tissue selected from Islets of Langerhans, and individual cells thereof, suspended in a physiologically compatible tissue medium.

25. The process of claim 13 wherein the core material comprises living tissue in a physiologically compatible tissue medium.

26. A process for producing a substance which is produced by living cells, the process comprising the steps of:

A. enclosing the cells within a permanent, covalently cross-linked semipermeable membrane having a selected upper limit of permeability, the enclosure being effected by the process of claims 1 or

13;

B. suspending the enclosed cells in an aqueous culture medium; and

C. allowing the cells to undergo metabolism in vitro and to produce the substance.

27. The process of claim 26 wherein the substance produced is insulin, glucagon, antibodies, prolactin, somatostatin, thyroxin, steroid hormones, pituitary hormones, interferons, FSH or PTH.

28. The process of claim 26 wherein the substance has a molecular weight below the selected upper permeability limit, the process comprising the step of allowing the substance to diffuse through the membranes into the aqueous medium, and harvesting the substance therefrom.

29. The process of claim 26 wherein the substance has a molecular weight above the selected upper permeability limit, the process comprising the step of separating the semipermeable membranes with enclosed cells and substance from the aqueous medium, and harvesting the substance therefrom.

30. An insulin producing unit comprising one or more viable, healthy, physiologically active mammalian Islets of Langerhans or cells therefrom enclosed within a space defined by a permanent, covalently cross-linked semipermeable membrane produced by the process of claims 1 or 13, the membrane being permeable to insulin produced by the Islets or cells therefrom, but impermeable to molecules having a molecular weight in excess of about 100,000 daltons and to viruses and bacteria.

31. An artificial organ suitable for implantation in a mammalian body comprising a permanent, covalently crosslinked semipermeable membrane, the membrane being prepared by the process of claims 1 or 13, and the membrane defining a space which encloses one or more viable, healthy, physiologically active cells or tissue, the membrane being impermeable to immune system mediators having a molecular weight in excess of about 100,000 daltons, but permeable to nutrients, hormones and other messenger molecules, and metabolic products produced by the cells or tissue.

32. The artificial organ of claim 31 wherein the tissue or cells comprise pancreatic endocrine cells, thymic cells, thyroid cells, liver cells, adrenal cells, blood cells, lymphoid cells or pituitary cells.

33. A cell or tissue implantation method comprising the steps of:

A. enclosing living cells or tissue within a space defined by a permanent, covalently cross-linked semipermeable membrane prepared by the process of claims 1 or 13, the membrane being impermeable to immune system mediators having a molecular weight in excess of about 100,000 daltons, but permeable to nutrients, hormones and other messenger molecules, and metabolic products produced by the cells or tissue, the cells or tissue after enclosure being viable, healthy, physiologically active cells or tissue capable of ongoing metabolism; and

B. introducing the enclosed cells or tissue into a mammalian body.

34. The implantation method of claim 33, wherein the tissue or cells comprise pancreatic endocrine cells, thymic cells, thyroid cells, liver

SUBSTITUTESHEET cells, adrenal cells, blood cells, lymphoid cells, or pituitary cells.

35. A process for culturing anchorage dependent cells, the process comprising the steps of: A. suspending cells in a medium containing an anchorage substrate material and hig molecular weight components needed to maintain viability and to support replication and/or differentiation of the cells;

B. enclosing the cells together with the medium and anchorage substrate material within a space defined by a permanent, covalently cross-linked semipermeable membrane produced by the process of claims 1 or 13, the semipermeable membrane having an upper limit of permeability sufficient to preclude traverse of the anchorage substrate material and sufficient to allow low molecular weight molecules to traverse the membrane;

C. suspending the product of step B in a culture medium sufficient to maintain viability and to support replication and/or differentiation of the encapsulated cells; and

D. allowing the cells to replicate and/or differentiate within the space defined by the membrane.