WO1993008766A1

WO1993008766A1 - Transplant protective coating

Info

Publication number: WO1993008766A1
Application number: PCT/US1992/007967
Authority: WO
Inventors: Kent Cochrum; Scott King
Original assignee: Transtech Medical, Inc.
Priority date: 1991-10-31
Filing date: 1992-09-25
Publication date: 1993-05-13
Also published as: IL103284A0; ZA927463B; AU2688392A

Abstract

Viable cells are coated with a neutral or charged biocompatible polymer impregnated with a substance which repels macrophages, lymphocytes or fibroblasts. The coated cells improve transplant viability.

Description

Transplant Protective Coating

Technical Field

The present invention relates to a coating for transplantation of tissues and cells and, in particular, to a coating for implantation of pancreatic islets.

Background Art

Transplants often fail due to induction of an immune reaction by the host against the implanted organ. The response can lead to rejection or destruction of the organ. Furthermore, if the transplanted organ contains immunocompetent cells, the organ can mount an immune reaction to the host, referred to as graft-versus-host disease.

Since the ability to closely match the host and donor organs is hampered by the limited availability of donor organs, a number of techniques have been devised to reduce the immunogenicity of transplants and to minimize the ability of the transplanted organ to mount an immune response.

One approach is to coat the transplanted cells with a membrane that minimizes the immunogenicity of the cells and/or protects the implant from the host. A variety of encapsulation methods have been used to apply a large number of different types of membranes with some success.

One of the problems with coating methods is that the tissue can be irregularly shaped or can have dead tissue clinging to the surface (acinar tissue) . A coating which is sufficiently thin to permit ingress and egress of nutrients and hormones may not be sufficiently thick to coat the entire surface of the transplant. In particular, this problem has been encountered with islet cells where accompanying acinar tissue leads to an irregular surface and can prevent complete encapsulation of the implanted cells.

Several methods for coating cells are known in the art. However, each of these methods fails to produce a satisfactorily coated cell, produces a coated cell which is immunogenic and subject to attack by the host immune system or becomes isolated by fibrosis, resulting in transplanted cell death shortly after transplantation. A summary of known encapsulation methods is described below.

Nilsson et al, Eur. J. Appl. Microbiol. Biotechnol. 47:319-326 (1983) describes a method of entrapping cells in agarose, agar, carrageenan, alginate, fibrin and polyacryla ide_

Nilsson et al, Nature, 302:629-630 (1983) describes encapsulation of animal cells that secrete large molecules such as virus vaccines, immunochemicals, hormones or enzymes in agarose beads. The beads are formed by cooling a cell-agarose suspension in an oil medium. Secretion of interleukin 2 and antibodies into the supernatant by the encapsulated cells was demonstrated.

U.S. Patent No. ^"4,352,883, and U.S. Patent No. 4,391,909 describe tissue cells, particularly islet of

Langerhans cells or liver cells , which are encapsulated in a spheroidal semipermeable polysaccharide membrane crosslinked with a polymer. The membrane is exemplified by sodium alginate that is crosslinked using polylysine.

U.S. Patent No. 4,673,566, U.S. Patent No. 4,689,293 and U.S. Patent No. 4,806,355 describe microencapsulation of living cells, particularly islet cells, for implantation using a semipermeable membrane exemplified by sodium alginate that is crosslinked using high molecular weight polylysine.

U.S. Patent No. 4,696,286 describes a coating for transplants, particularly pancreatic islets. The coating is formed from a nontoxic inner layer which is bonded to an outer biologically compatible layer, which may be comprised of a plurality of coats of the same or different polymers. The coating can also include an intermediate layer of an innocuous material.

The inner layer is comprised of aluminum hydroxide, a disaccharide (such as maltose, sucrose and lactose) , a polyfunctional crosslinking agent (such as 3,3'- di ethyldithiobispropionate) , an immunoglobulin to a surface component of the transplant (such as antibodies to MHC antigens) , a lectin (such as concavalin A) , or a polyionic polyamino acid (such as polylysine) . The inner layer is bound to an outer, semipermeable, biologically compatible layer (such as polylysine and other polyamino acids) .

U.S. Patent No. 4,789,550 describes spherical, smooth and uniform microcapsules which are suitable for cardiovascular injection and may contain living cells, particularly pancreatic islets, and a method of making the capsules. The semipermeable, biocompatible capsules may be directly injected into the bloodstream so that they lodge inside organs such as the liver and spleen where they are washed by blood. The capsules are preferably made by forming temporary capsules of a polysaccharide gum, most preferably sodium alginate, which is crosslinked or hardened by biocompatible polymers, most preferably polylysine.

International application Serial No. PCT/US88/02413 (Publication No. 89/01034) describes a method for encapsulating a biological material such as viable cells in a semipermeable membrane which permits entry of nutrients and prevents entry of high molecular weight substances such as antibodies, toxins and bacteria. The material to be encapsulated is suspended in a medium containing an effective amount of a gelling inducer and the suspension is formed into a droplet of a sufficient size to envelop the material. Then a discrete capsule is formed by contacting the outer surface portion of the droplet with a gelling solution containing an effective amount of a gel forming polymer which gels upon contact with the gelling inducer. The encapsulated materials remain in the original suspension environment which never comes into contact with the gel forming polymer.

Disclosure of the Invention

Accordingly, one object of the present invention is to provide a method for coating viable cells, preferably endocrine cells such as pancreatic islet cells, which provides coated cells which are not immunogenic in the host organ and may be vascularized to provide implanted cells having an improved implant lifetime.

This and other objects of the present invention which will become apparent from the following description have been achieved by the present improved coating method and coated transplant cells.

The present invention provides an improved coating for transplants comprising a neutral or charged biocompatible polymer impregnated with a substance which repels macrophages, lymphocytes, and/or fibroblasts. The repellent is compatible with transplanted cell viability and function. Preferred repellents include soluble and slightly soluble antiinflammatory corticosteroids, immunosuppressive drugs, biological response modifiers, hormones, non-steroidal anti-inflammatory drugs, and whole antibodies or Fab fragments thereof which bind selectively with HLA Class I and Class II antigens. Use of the repellent in the coating increases transplant viability.

Best Mode for Carrying Out the Invention

The present method can be used on any tissues and cells used in medical transplantation. Tissues and cells which may be transplanted include cells from endocrine tissue such as adrenal gland cells, pituitary gland cells and pancreatic islet cells, and liver cells (hepatocytes) , etc as well as other viable organ cells. Other cells such as yeast cells, algal cells, plant cells, bacteria, etc., may also be encapsulated. Further, hybridoma cells which have been transformed by standard genetic engineering techniques to enable the hybridoma cells to produce large quantities of a specific protein product, may be encapsulated. The encapsulated hybridoma cells may then be used to produce phar acologic amounts of the desired protein product at the site of transplantation. The method is particularly advantageous for implantation of pancreatic islets.

The isolation method of the cells does not affect the encapsulation method of this invention. The cells will usually be a digest prepared by a mechanical means and, more usually, also with an enzymatic digestion method that produces single cells and cell aggregates. Preferably, the selected method will minimize the amount of dead tissue and tissue debris present in the isolated cells. Methods of isolating individual cells and cell aggregates are well known in the art. Any of these known methods for producing cells or cell aggregates may be used in the present invention.

The cells or cell aggregates prepared as described above are then coated with the desired coating material containing a repellent. The repellent is mixed with the coating materials prior to application of the coating to the isolated cells. The repellent can be added to each of the coating layers, or can be added only to the outer layer or layers of multiple-layer coatings. Thereafter, the coatings are applied to the isolated cells in the same manner as known in the art. That is, the addition of the repellent to the coating layers does not affect the method by which the coating is applied. In addition, the use of the repellent does not necessitate any changes in the way the coating solutions are prepared.

The coatings may be prepared from any of the known coating materials which are compatible with the cell type to be implanted and the repellent used. Numerous implantation coatings are well known. Suitable coatings include alginate, agar and agarose. A description of the preparation of exemplary coatings is provided below. A preferred coating is a polysaccharide-based, preferably an agarose-based coating. If desired, the coating may further contain a bonding layer, a bridge layer and an outer layer. In this embodiment, the bridge layer is preferably impregnated with the repellent.

Polysaccharide gums suitable for coating cells for implantation include alkali metal alginates, guar gum, gum arabic, xanthan gum and acidic fractions thereof. The polysaccharide gum coatings are generally prepared by mixing the tissue to be coated in a physiological solution such as saline, phosphate buffered saline (PBS) or a nutrient medium with the water-soluble gum. The gum-tissue suspension is formed into droplets. The droplets are solidified by cooling below the gelation temperature with agarose, for example, or by addition of a gel inducer such as calcium chloride with alginate.

For sodium alginate a solution of sodium alginate is mixed with the desired cells (Eur. J. Appl. Microbiol. Biotechnicol. , 417:319-326 (1983)). Droplets are formed by suspending the mixture in a hydrocarbon phase such as soy, paraffin or silicon oil which is compatible with the cell type and stirring until a desired droplet size is produced. This mixture is rapidly mixed with an excess of CaCl₂ solution. The beads are allowed to stabilize and then collected using a nylon net. The encapsulated cells are suitable for implantation at this point or can be crosslinked as described below.

U.S. Patent Nos. 4,352,883 and 4,391,909 describe use of 1.0 to 2.0% solution of gum, preferably 1.0 to 1.5% for sodium alginate. The gum solution/cell suspension is formed into droplets by forcing the suspension through a vibrating capillary tube placed in the center of a vortex created by rapidly stirring an aqueous solution containing a multivalent cation, preferably 1.5% CaCl₂. Droplets released from the tube into the gel inducer form spherical particles.

Other methods of generating droplets of the appropriate size and shape are described in U.S. Patent Nos. 4,673,566, 4,689,293, 4,806,355 and 4,789,550. The methods include use of a droplet generating device. These references are incorporated herein by reference for a more complete description of available methods and materials.

In some instances, improved results have been achieved by coating the particles with a polymer having charged groups. Preferred polymers are polyamino acids such as polyarginine, most preferably poly-L-lysine. Other charged polyamino acids such as polyaspartic or polyglutamic acid are also suitable. The crosslinking polymer is used as a dilute solution (from about 0.01 to 1.0 wt.%) in a physiological solution, (a.g., normal saline, and is mixed with the particles for from at least 3 and preferably about 3 to about 10 minutes depending on the degree of rigidity desired. The molecular weight of the polymer can vary from about 3,000 to 500,000 daltons or higher, depending on the desired stability and permeability of the encapsulated cells.

Agarose coatings may be prepared using a warm agar or agarose solution mixed with the cells to be coated. Droplets are formed by suspending the mixture in a hydrocarbon phase such as soy, paraffin or silicon oil which is compatible with the cell type and stirring until a desired droplet size is produced. This mixture is cooled in an ice bath with continued stirring until the polymer solidifies. The mixture is then transferred to centrifuge tubes to which water, buffer or medium is added. The beads are spun down and the aqueous phase is removed.

Polymer coatings having surface charge may also be used to prepare the coatings of the present invention. An im unological barrier membrane which conforms to the surface of a transplant material and contains an inner layer that is bonded to the surface of the transplant material and an outer biologically compatible, water insoluble semipermeable layer bonded to the inner layer is described in U.S. Patent No. 4,696,286 and may be used in the present invention.

The non-cytotoxic inner layer may be aluminum hydroxide, a disaccharide (such as maltose, sucrose and lactose) , a polyfunctional crosslinking agent (such as 3,3'-dimethyldithiobispropionate) , an immunoglobulin to a surface component of the transplant (such as antibodies to KHC antigens) , a lectin (such as concavalin A) , or a polyionic polyamino acid (such as polylysine) . The inner coating is chemically bound to an outer, semipermeable, biologically compatible layer (such as polylysine or other polyamino acids) .

The repellant-containing coating of the present invention may also be applied using the method described in International application Serial No. PCT/US88/02413 (Publication No. 89/01034) . The material to be encapsulated is suspended in a medium containing an effective amount of a gelling inducer. The suspension is formed into a droplet of a sufficient size to envelop the cells. Then, a discrete capsule is formed by contacting the outer surface portion of the droplet with a gelling solution containing an effective amount of a gel forming polymer which gels upon contact with the gelling inducer. The encapsulated materials remain in the original suspension environment and do not come into contact with the gel forming polymer.

The cells are suspended in a suitable aqueous medium such as a physiological buffer solution. The buffer contains an amount of a gelling inducer sufficient for the inducer to differ outwardly to contact the gel forming polymer and induce polymerization. The aqueous medium may also contain a viscosity enhancer such as dextran, hyaluronic acid, polyethylene glycol or starch.

The cell suspension is formed into droplets of a sufficient size to envelope the cells by conventional methods; e.g., by using a nozzle, capillary tube or hypodermic needle. The droplet of the formed suspension is dropped into a rapidly stirring solution of the gel forming polymer. The contact forms a gel around the outer surface of the droplet. The gel forming polymer is preferably a polysaccharide such as sodium alginate, guar gum, gum arabic, carrageenan, pectin, tragacanth gum, xanthan gum, or deacylated chitin (chitosan) . The resultant capsules can be recovered and equilibrated with the desired implantation medium.

The application describes a number of alterations that can be used to adapt the capsule for particular purposes. These alterations may also be used in the present invention. In particular, a second gel forming polymer can be used to impart altered properties to the membrane such as mechanical strength, chemical stability, pore, size and/or surface charge. The second polymer is preferably a polyelectrolyte having a charge opposite to that of the first polymer, for example, polylysine. Alternate embodiments include application of a second coating layer which reacts with the first coating layer.

In a preferred embodiment of the present invention, a three part coating is used to encapsulate the transplanted cells coating more effectively than the previously described coatings. The inner, bonding layer is of positively-charged, polyionic polymer which coats the transplant, providing a substantially uniform surface charge. The intermediate bridge layer is a polysaccharide, preferably also containing a negatively-charged polyamino acid, and provides a uniformly smooth shape to the transplant. The outer layer is a positively-charged polyionic polymer that crosslinks the bridge layer, providing a more stable coating.

Biological membranes generally exhibit a negative charge on the membrane surface. A positively-charged polyionic polymer, preferably poly-L-lysine, is therefore used as the inner bonding layer to coat the cells. The inner bonding layer is charge attracted to the biological membrane and provides a substantially uniformly charge on the surface of the transplant for effective coating by the intermediate layer. The inner layer may be relatively thin and still insure effective coating by the intermediate layer.

The positively-charged polyionic polymer may be any positively-charged, polyionic polymer which is compatible with cell viability. Numerous positively-charged polyionic polymers are well known to be suitable for encapsulation purposes. Preferably, the positively-charged polyionic polymer is a polyamino acid, most preferably polyarginine or polylysine. The molecular weight of the polymer may vary from about 15,000 to 300,000 daltons or higher, but is preferably 15,000 to 22,000 daltons. Increases in the chain size of the polymer produce increased pore size.

The inner bonding layer is formed by combining cells with a dilute solution (from about 0.01 to 1.0 wt.%) of the polyionic polymer in a physiological solution, e.g., normal saline for from about 1 to about 10 minutes to coat the cells to form particles. The time depends on the selected polymer, the molecular weight of the polymer and the temperature. The longer the time, the smaller the resultant pore size.

The intermediate bridge layer coats the transplanted cells, forming a substantially uniform surface by filling in irregular surfaces and coating any attached tissue and debris. The bridge layer is formed of a polysaccharide, most preferably agarose, and has a charge opposite to that of the bonding layer. In a preferred embodiment, the layer also contains the repellent and a negatively charged polyionic polymer, preferably a polyamino acid, to facilitate joining the bridge layer to the bonding layer. The bonding layer and intermediate bridge layer can be added at one time if polylysine is added to the agarose at a concentration of from about 0.01 wt.% to about 1.0 wt.%, preferably about 0.5 wt.%.

Suitable polysaccharides include agar, carrageenan, purified sodium alginate, and agarose. Agarose is most preferred due to its biocompatibility. The polysaccharide is generally used at a concentration of from about 0.5 wt.% to about 6 wt.% in a physiological solution such as normal saline. The bridge layer may also contain a negatively charged polyionic polymer, preferably a polyamino acid, most preferably polyglutamic acid or polyaspartic acid. The molecular weight of the polyionic polymer may vary from about 3000 to 500,000 daltons or larger, depending on the desired pore size of the membrane. The ratio of polyglutamic acid to agarose is from about 0.01 wt.% to about 1.0 wt.%, preferable about 0.5 wt.%.

The bridge layer containing the repellent is added to the coated cell particles by stirring the particles in oil and adding a solution of the bridge layer materials. This -13-

coating method is well known and is referred to as the "water and oil" method. Briefly, at room temperature, the particles and coating mixture are dropped into a nontoxic oil, e.g., a silicone oil, while stirring the oil. When 5 the desired particle size is achieved, the particles are removed and rinsed in a suitable buffer, e.g. PBS. About 1 to about 10 minutes, preferably 1 to 6 minutes, is usually sufficient to form a bridge layer about 10 microns to 300 microns in size. That thickness is usually sufficient to 10 provide a regular surface to any type of cell implant preparation. The bridge layer can consist of multiple coating layers.

The outer layer is formed by crosslinking the bridge layer using a crosslinking agent having a charge opposite

15 to that of the intermediate layer. The positively charged, polyionic polymer may be any positively charged, polyionic polymer which is compatible with cell viability. The outer layer is preferably formed from positively charged polyamino acid. Numerous positively charged polyionic

20 polymers are well known to be suitable as crosslinking agents for encapsulation purposes. Polylysine is most preferred since it minimizes fibrosis upon implantation. The molecular weight of the polymer may vary from about 3,000 to 10,000 daltons or higher, depending on the degree

25 of rigidity and permeability desired.

The outer layer is formed by combining the two layer- coated cell particles with a dilute solution (from about 0.01 to 0.05%) of polyionic polymer in a physiological solution, e,g., normal saline for from about 1 to about 10 30 minutes to coat the cells to form the particles for implantation.

The concentration and duration of the reaction, together with the molecular weight of the polymer, determine the degree of permeability of the particle and may be routinely varied to achieve the desired permeability. To some extent, the amount of crosslinking determines the length of time the particle remains protected following implantation.

The repellent is defined as a substance which repels a macrophage, lymphocyte or fibroblast, keeping it away from the transplant. The repellent must be compatible with the viability and function of the encapsulated cell. Preferred repellents include soluble and slightly soluble anti- inflammatory corticosteroids, immunosuppressive drugs, biological response modifiers, hormones, nonsteroidal anti- inflammatory drugs whole antibodies or Fab fragments thereof which bind selectively with HLA Class I and Class II antigens. Use of the repellent in the coating increases transplant viability.

A single repellent can be used in each coating, however, combinations of-repellents are also contemplated. Usually, when repellents are combined, the repellents are from different categories. That is, the combination usually includes, for example, an immunosuppressant together with a biological response modifier, rather than two immunosuppressants. Of course, some repellent agents may properly be classified in more than one category. For example, some biological response modifiers may also be hormones. In such cases, two agents from the same category may be used. The effective concentration of a repellent may vary upon combination with another repellent as is well known.

The repellent may be used in any of the coating layers in any of the methods described above by combining the repellent with the coating layer material prior to coating. The repellent must be present at a concentration which allows for gradual diffusion of the repellent out of the coated implant for a predetermined time period thereby repelling macrophages, lymphocytes and fibroblasts.

Generally, the repellent is incorporated into a coating material in a weight ratio of from about 10^"12 to 10^"2 of the weight of the coating material, depending upon the repellent and its relative potency. When combinations of repellents are used, both repellents will generally be present in amounts within the general concentration range. Specific concentrations for individual repellents can be readily determined by one having ordinary skill in the art and will depend upon the specific combination of repellents used and the desired lifetime of the transplant.

Preferred soluble and slightly soluble anti-inflammatory corticosteroids include hydrocortisone, prednisolone, cortisone, prednisone, dexamethasone, fluocinolone acetonide, flumethasone, fluocortolone, methylprednisolone, meprednisone, triamcinolone, fluprednisolone, betamethasone, etc.

Preferred immunosuppressive drugs include cyclosporin, FK506, rapamycin, azathioprine, cyclophosphamide, etc. Preferred biological response modifiers include macrophage inhibitor factor, superoxide dismutase, fibroblast growth factor, etc.

In addition to the above described corticosteroids, other hormones such as antiphlogistic prostaglandins such as PGE and PGE₂, for example, can be used.

Suitable nonsteroidal anti-inflammatory drugs include prostaglandin inhibitors such as sodium salicylate, ibuprofen, naprosyn, indomethacin, phenylbutazone, piroxica, aspirin and lazaroids.

Any of the repellents or combinations thereof may be mixed with the coating materials prior to coating of the transplant cells. The repellent is added to and mixed into the coating material using any well known stirring means. Preferably, the stirring is vigorous so as to form a uniform concentration of the repellent throughout the coating. For example, when coating with sodium alginate, the desired repellent is added to the sodium alginate solution prior to droplet formation. Similarly, the repellent may be added to coating solutions of polyamino acids, agarose and aluminum hydroxide prior to coating. In preferred embodiments, the repellent is not added to the final outer membrane. In these embodiments, .the outer membrane functions to limit diffusion of the repellent away from the transplant thereby affecting the time-release characteristics of the repellent from the transplant. Adjustment of the pore size of the outer membrane using the methods described above allows one to control and adjust the time course of the repellent release from the transplant. Specific time-release profiles can be readily obtained by one having ordinary skill in the art using known methods of adjusting the pore size of the membrane for a given repellent.

This invention is further illustrated by the following specific but non-limiting examples. Temperatures are given in degrees Centigrade and concentrations as weight percent unless otherwise specified. Procedures which are constructively reduced to practice are described in the present tense, and procedures which have been carried out in the laboratory are set forth in the past tense. EXAMPLES

Example 1

This example illustrates the microencapsulation of islets of Langerhans. Cultured rat islets of Langerhans (2 X 10³ islets in 0.2 ml medium) are suspended uniformly in 2 ml of a 1.5% (w/w) sodium alginate solution in physiological saline. Spherical droplets containing islets are produced by syringe pumper jet extrusion through a 22-gauge needle and collected in 1.5% (w/w) calcium chloride solution. The supernatant is decanted and the gelled spherical alginate droplets, containing islets, are washed with dilute CHES (2-cyclohexylaminoethane sulfonic acid) solution and 1.1% calcium chloride solution.

After aspirating off the supernatant, the gelled droplets are incubated for 6 minutes in 0.05% (w/w) polylysine having a molecular weight of 17,000 containing 0.1 wt.% hydrocortisone.

The supernatant is decanted and the polylysine capsules are then washed with dilute CHES, 1.1% calcium chloride solution and physiological saline. The washed polylysine capsules are incubated for 4 minutes in 30 ml of 0.03% sodium alginate to permit the formation of an outer alginate membrane on the initial polylysine membrane, by ionic interaction between the negatively charged alginate and the positively charged polylysine.

The resulting microcapsules are washed with saline, 0.05M citrate buffer for 6 minutes to reliquify the inner calcium alginate, and a final saline wash is performed. The microcapsules are found to be spherical and each to contain from 1 to 2 viable islets. When the experiment is repeated with islet cells from mouse, bovine and dog pancreas and similar microencapsulated products are formed.

Example 2

Islets of Langerhans are obtained from rat pancreas and added to a complete tissue culture medium at a concentration of approximately 10³ islets per milliliter. The tissue culture medium contains all nutrients needed for continued viability of the islets as well as the amino acids employed by the beta cells for making insulin.

Fourteenths of a milliliter of the islet suspension are then added to a one-half milliliter volume of 1.2 percent sodium alginate (Sigma Chemical Company) in physiological saline.

Next, 80 milliliters of a 1.5 percent calcium chloride solution are placed in a 150 milliliter beaker on a stirrer and stirred at a rate which induced the formation of a vortex having a conical-shaped void at its center. A glass capillary having a gradually decreasing diameter ending in a tip of inside diameter about 300 microns is then fitted with a vibrator at 60 cycles per second. The capillary tip is then placed within the center of the vortex, the vibrator turned on and the sodium alginate-culture medium-tissue suspension is formed therethrough with an infusion pump. Droplets on the order of 300-400 microns in diameter are thrown from the tip of the capillary and immediately enter the calcium solution.

After 10 minutes, the stirrer is turned off and the supernatant solution is removed by aspiration. The gelled capsules are then transferred to a beaker containing 15 mol of a solution comprising one part of a 2% 2-(cyclohexylamino) ethane sulfonic acid solution in 0.6% NaCl (isotonic, pH=8.2) diluted with 20 parts 1% CaCl₂- After a 3 minute immersion, the capsules are washed twice in 1% CaCl₂.

The capsules are then transferred to a 32 ml solution comprising 1/80 of one percent polylysine (average MW 35,000) and 0.1 wt.%, 5 wt.% or 10 wt.% separately of one of dexamethasone, cyclosporin, ibuprofen or naprosyn in physiological saline. After 3 minutes, the polylysine solution is decanted. The capsules are then washed with 1% CaCl₂, and then suspended for 3 minutes in a solution of polyethyleneimine (MW 40,000-60,000) produced by diluting a stock 3.3% polyethyleneimine solution in morpholinopropane sulfonic acid buffer (0.2 M, pH-6) with sufficient 1% CaCl₂ to result in a final polymer concentration of 0.12%. The resulting capsules, having permanent semipermeable membranes, are then washed twice with 1% CaCl₂, twice with physiological saline, and mixed with 10 ml of a 0.12 percent alginic acid solution.

The capsules resist clumping, and many can be seen to contain islets of Langerhans. Gel on the interior of the capsules is reliquified by immersing the capsules in a mixture of saline and citrate buffer (pH=7.4) for 5 minutes. Lastly, the capsules are suspended in CMLR-69 medium.

Example 3

Fresh pancreatic tissue is comminuted and placed in Hank's solution containing collagenase to digest connective tissue. The resulting digest is subjected to Ficoll-Hypaque gradient centrifugation to isolate the islets. The isolated islets are cultured for 7 days at 37°C in RPMI 1640 medium supplemented with lot fetal calf serum under a moist 5% C0₂ atmosphere.

Example 3a:

Isolated islets are suspended in 3 ml RPMI 1640 at a concentration of 10³ islet per ml. Aluminum hydroxide

(Sigma Chemical Company) is ground in a mortar and pestle until the gel particle size is 1-3 microns. A one percent AL(OH)₃ solution is made in physiological saline. The RPMI medium is removed from the islet and replaced with 3 ml of the 1% AL(OH)₃ saline solution. The islet-Al(OH)₃ solution is mixed by rotation for 2.5 min. The Al(OH)₃-coated islets are sedimented out and the excess Al(OH)₃ solution removed. The coated islets are then washed 3 times in 6 ml physiological saline, pH 7.

The coated islets are then transferred to 3 ml of a

0.5% physiological saline, pH 7 solution of poly-L-aspartic acid, MW 50,000 (Sigma Chemical Company) containing 1.0 wt.% indomethacin and mixed for 4 min. The poly-L-aspartic is removed and the coated tissue islets washed 3 times with 6 ml of physiological saline, pH 7.

The coated islets are then suspended in 3 ml of 0.5% solution of poly-L-lysine MW 50,000 (Sigma Chemical Company) and mixed for 5 min. The poly-L-lysine is removed and the islets are washed 3 times in physiological saline, pH 7.

The poly-L-aspartic acid and poly-L-lysine coatings and washes may be repeated if a thicker outer layer is desired.

Following the final physiological saline wash the coated islets are suspended in 10 ml of a 1% solution of deferoxamine (Ciba-Geigy) in physiological saline, pH 7.2 for 10 min. The deferoxamine treatment is repeated another 10 min. and then removed. The coated islets washed 2 times in physiological saline and RPMI media. The islets can be transplanted at this point returned to tissue culture. The coated islets can be obtained in tissue culture in RPMI 1640, 10% fetal serum, 5% C0₂, 85% air.

Example 3b:

Isolated islets are suspended in 3 ml of a 0.5% physiological saline, pH 7, solution of poly-L-lysine, MW 50,000, and mixed for approximately 10 min. The poly-L- lysine solution is then removed and the coated islets washed 3 times with 6 ml of physiological saline.

The coated islets are then transferred to 3 ml of a

0.5% physiological saline solution of poly-L-aspartic acid, MW 50,000 containing 0.1 wt.% prednisone, and mixed for approximately 10 min. The poly-L-aspartic is removed and the coated islets are again washed 3 times with saline.

Finally, the coated islets are again suspended in 3 ml of the 0.5% saline solution of poly-L-lysine, and mixed for approximately 10 min. followed by washing in saline.

Example 4

Pancreatic islets are dispersed in 3 ml of a 2 wt.% solution of agarose in physiological saline, pH 7, containing 0.1 wt.% dexamethasone acetate and 0.5 wt.% polylysine (MW 20,000 daltons) and mixed for 10 min.

Spherical droplets containing islets are produced by syringe pump jet extrusion through a 22 gauge needle and collected in cold saline (4-10°C) .

The supernatant is decanted, and the gelled spherical agarose droplets containing islets are washed with saline.

After aspirating off the supernatant, the gelled spheres are incubated for 6 min in 3 ml of physiological saline, pH 7, containing 0.5 wt.% poly-L-aspartic acid or 0.2 wt.% sodium alginate. The poly-L-aspartic acid or sodium alginate solution is removed, and the spheres are rinsed with saline. The coated islets are then coated by treating them with an aqueous solution containing 0.5 wt.% poly-L-lysine (MW 20,000 daltons) for 5 min., removing them from the poly-L-lysine solution and washing them with saline.

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims

1. A transplant having an immunological barrier coating, comprising:

an immunological barrier coating enclosing a transplant material, said coating containing a repellently effective amount of at least one of a macrophage, lymphocyte, or fibroblast repellent.

2. The transplant of Claim l, wherein said transplant comprises endocrine cells.

3. The transplant of Claim 1, wherein said transplant comprises pancreatic islet cells.

4. The transplant of Claim l, wherein said coating comprises a weight ratio of repellant to coating material of from 10^"12 to 10^'2.

5. The transplant of Claim 1, wherein said immunological barrier coating comprises agarose.

6. The transplant of Claim 1, wherein said immunological barrier coating comprises two layers, an inner layer bonded to the surface of the transplant material and an outer biologically compatible layer.

7. The transplant of Claim 6, wherein said inner layer comprises agarose.

8. The transplant of Claim 1, wherein said immunological barrier membrane comprises multiple alternating layers of positively-charged and negatively-charged polymers, wherein at least one of said layers contains repellent.

9. The transplant of Claim 8, wherein said positively-charged and negatively-charged polymers are polyamino acid polymers.

10. The transplant of Claim 9, wherein said polyamino acid polymers are selected from the group consisting of polylysine, polyglutamic acid, polyarginine and polyaspartic acid.

11. A method of encapsulating a transplant for transplantation, comprising the steps of:

coating a transplant material with an immunological barrier coating containing a repellently effective amount of a macrophage, lymphocyte, or fibroblast repellent.

12. The method of Claim 11, wherein said transplant comprises endocrine cells.

13. The method of Claim 11, wherein said transplant comprises pancreatic islet cells.

14. The method of Claim 11, wherein said coating contains a weight ratio of repellent to coating material of 10^"12 to 10^"2.

15. The method of Claim 11, wherein said immunological barrier membrane-comprises agarose.

16. The method of Claim 11, wherein said immunological barrier coating comprises two layers, an inner layer bonded to the surface of the transplant material and an outer biologically compatible layer.

17. The method of Claim 16, wherein said inner layer comprises agarose.

18. The method of Claim 11, wherein said immunological barrier coating comprises multiple alternating layers of positively-charged and negatively-charged polymers wherein at least one of said layers contains repellent.

19. The method of Claim 18, wherein said positively- charged and negatively-charged polymers are polyamino acid polymers.

20. The method of Claim 19, wherein said polyamino acid polymers are selected from the group consisting of polylysine, polyglutamic acid, polyarginine and polyaspartic acid.