CA1145258A

CA1145258A - Encapsulation of viable tissue and tissue implantation method

Info

Publication number: CA1145258A
Application number: CA000348524A
Authority: CA
Inventors: Franklin Lim
Original assignee: Damon Corp
Current assignee: Damon Corp
Priority date: 1979-03-28
Filing date: 1980-03-26
Publication date: 1983-04-26
Also published as: BE882476A; CH653914A5; US4352883A; SE8002357L; NO160380B; NO800901L; IT1133081B; CH657786A5; DE3012233C2; JPS55157502A; NO160380C; JPS6239131B2; FR2457688A1; IT8067472A0; FR2452285A1; JPS61293919A; DE3012233A1; GB2046209B; JPS6242889B2; SE448060B

Abstract

ABSTRACT OF THE DISCLOSURE
Viable tissue such as mammalian islets of Langerhans or liver tissue is encapsulated within a semipermeable membrane which allows the passage of oxygen, amino acids and nutrients needed for maintenance and ongoing metabolism of the tissue, but is impermeable to bacteria, lymphocytes, and proteins having a molecular weight above a selected level so as to exclude poten-tially deleterious large molecules such as immunoglobulins. By means of the process, and insulin generating system can be pro-duced wherein mammalian islets are maintained in a viable and protected state within the capsule and excrete hormones. Also, mammalian liver tissue can be encapsulated for use as a body fluid detoxification system. The encapsulated liver tissue is shielded from plasma protein of the type responsible for immuno-logical rejection and from leukocytes and bacteria, but toxins and nutrients required for maintenance and continued normal meta-bolism of the tissue freely traverse the membrane. The capsules may be injected into a suitable site in a mammalian body and act as an artificial organ, e.g., and artificial pancreas. The pro-cess comprises the steps of suspending the material to be encap-sulated in a physiologically compatible medium such as a culture medium containing a water soluble substance which can be gelled into droplets to form discrete, shape-retaining temporary cap-sules, forming a semipermeable membrane about the temporary cap-sules, and optionally, reliquify the gelled interior, The pro-cess can also be used for encapsulating chemically active sub-stances such as activated charcoal particles and labile biolog-ical materials.

Description

~5~S~ ~' I BACK~:ROUND OF THE INVENTION
This invention relates to a process for encapsulat-ing 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 maintain the -tissue and support its normal metabolic functions, but impermeable to bacteria, lymphocytes, and large proteins of the type responsible for immunochemical reactions re-sulting in rejection. The process enables the production of, for example, an insulin producing system or other hormone producing system as it allows encapsulation of mammalian pancreatic beta cells, alpha cells, intact islets of Langerhans, and other tissues or tissue fractions which secrete hormones. The capsules may be suspended in a culture medium and will excrete hormone over an extended period.
The capsules may also be used as an artificial pancreas hich can be implanted, e.g., by injection, into a diabetic mammal and wi]l function in vivo to excrete insulin and other hormones :in response to ambient sugar concentration.
It is believed that the art is devoid of methods for encapsulating living tissue such that the tissue remains viable. Attempts to accomplish this are frustrated by the conditions required for capsular membrane formation which are typically hostile to living systems. Canadian patent 1,~75,179 (Fi~m et al) discloses a technique for encapsulatin~ labile biological materials within asemip~able semipermeable .

1 membrance. This technique is capable, for example, of encap-sulatlng enzymes within a membrance from which the enzyme can-not escape, while allowing free passage of the enzyme's substrate ~owever, while the technique involves reaction conditions ~hich preserve the fragile operability of biological m~teri~ls~ no su-ggestion is made that living tissue can be encapsulated, Encapsulated live cells, organelles, or tissue have many potential uses. For example, within a semipermeable mem-brane, the encapsulated living material can be preserved in a permanent sterile environment and can be shielded from direct contact with large, potentially destructive molecular species,yet will allow free passage of lower molecular weight tissue nutrients and metabolic products. Thus, the development of such an encapsulation technique could lead to systems for producing useful hormones such as insulin. In such systems, the mammalian tissue responsible for the production of the material would be encapsulated in a manner to allow free passage of nutrients and metabolic products across the membrane, yet prohibit the passage of bacteria. I membrane permeability could be controlled~ it ~ is possible that this approach could also lead to artificial org~ns which could be implanted in a mammalian body, e.g., a diabetic, without rejection and with controlled hormone release, e g., insulin release triggered by glucose concentration.
Various attempts have been made to produce artificial organs suitable for implantation in mammalian bodies by providin~
a mechanical semipermeable barrier, e.g., a Millipore diffusion chamber or a capillary tube chamber, about tissue e~cised from a donor. Such artificial organs normally require surgical implantation. Furthermore, the protective mechanisms of mamMa]ian bodies isolate the implant, typically by plugging pores by fibroblastic overgrowth.

~s~

1 SUM~RY OF THE INVENTION
- In one aspect, the instant invention provides a method of eneapsulating core materials sueh as living tissue, individ-ual cells, or biologically active materials in a semipermeable membrane. The basic approach in~olves suspending the tissue to be eneapsulated in a physiologieally eompatible medium eon-taining a water soluble substanee that ean be maae insoluble in water, that is, gelled, to provide a temporary protective envi--ronment for the tissue. The medium is next formed into droplets eontainin~ the tissue and gelled, ~or example, by changing con-ditions of temperature, pH, or ionic environment. The "tempor-ary capsules" thereby produced are then sub~ected to a treatment, which ean be a known treatment, that results in the produetion ; of membranes of a eontrolled permeability (including impermea-bility) about the shape-retaining temporary eapsules.
~ The temporary capsules ean be fabrieated from any non--; toxie, water soluble substance that c:an be gelled to form a shape-retaininy mass by a change of eonditions in the medium in whieh it is placed, and also eomprises plural groups which are readily ionized to form anionie or eationic yroups. The pre-senee of such groups in the polymer enables surfaee layers of the capsule to be cross-linked to produee a "permanent" memhrane when exposed to polymers containing multiple funetionalities of the opposite charge.
The presently preferred material for forming the -tem-porary eapsules is polysaccharide gums, either natural or synthe-tie, of the type which can be a) gelled to form a s'nape-retaining mass by being exposed to a ehange in conditions sueh as a pH

ehange or by being exposed to multivalent eations sueh as Ca+~-;

and b) permanently "erosslinked" or hardened by polymers con-5~5~

1 taining reactive groups such as amine or imine groups which canreact with acidic polysaccharide constituents. The presently preferred gum is alkali metal alginate. Other water soluble gums which may be used include guar gum, gum arabic, carrageenan, pectin, tragacanth gum, xanthan gum or acidic fractions thereof~
I~hen encapsulating thermally refractory materials, gelatin or agar may be used in place of the gums.
The preferred method of formation of the droplets is to force the gum-nutrient-tissue suspension through a vibrating capillary tube placed within the center of the vortex created by rapidly stirring a solution of a multivalent cation. Droplets ejected from the tip of the capillary immediately contact the solution and gel as spheroidal shaped bodies.
The preferred method of forming a permanent semiper-meable membrane about the temporary capsules is to "crosslink"
surface layers of a gelled gum of the tpye having free acid groups with polymers containing acid reactive groups such as amine or imine groups. This is typica]ly done in a dilute solu-tion of the selected polymer. Generally, the ]ower the molecular ~ weight of the polymer, the greater the penetration into the sur-face of the temporary capsule, and the greater the penetration, the less permeable the resulting membrane. Permanent crosslinks are produced as a consequence of sa]t formation bet~een the acid reactive groups of the crosslinking polymer and the acid groups of the polysaccharide gum. Within limits, semipermeability can be controlled by setting the molecular weight of the crosslinking polymer, its concentration, and the duration of reaction.
Crosslinking polymers which have been used with success include polyethyleneimineand polylysine. Molecular weight can ~ary, depending on the degree of permeability required, between about ~5~58 1 3,000 and 100,000 or more. Good results have been obtained using polymers having an averag~ molecular weight on the order of 35,000.
The capsules can be engineered to have a selected in vivo useful life by astute selection of the cross-linking polymer. Proteins or polypeptide crosslinkers, e.g., poly-lysine, are readily attacked in vivo resulting in relatively rapid destruction of the membrane. Cross-linkers not readily digestible in mammalian bodies, e.g., polyethyleneimine, result in longer lasting membranes. By selecting the crosslinking polymer or by cross-linking simultaneously or sequentially with two or moxe such materials, it is possible to preselect the length of time the implanted tissue remains protected.
Optionally, with certain materials used t~ form the temporary capsules, it is possible to improve mass transfer within the capsule after formation of the permanent membrane by re-establishing the conditions under which the material is liquid, e.g., removing the multivalent cation. This can - be done by ion e~change, e.g., immersion in phosphate buffered saline or citrate buffer. In some situations, such as where it is desired to preserve the encapsulated tissue, or where the temporary gelled capsule is permeable, it may be preferable to leave the encapsulated gum in the crosslinked, gelled state.
An al'ternative method of membrane formation involves an interfacial polycondensation or polyaddition similar to the procedure disclosed in Canadian patent 1,075,179. This approach involves preparing a suspension of temporary capsules in an aqueous solution of the water soluble reactant of a pair of complemen-tary monomers which can form a polymer. Thereafter, the aqueous phase is suspended in a hydrophobic liquid in which ~s~

1 the complementa~y reactant is soluble. When the second react-ant is added to the two-phase system, polymerization takes place at the interface. Permeability can be controlled by controlling the ma~eup of the hydrophobic solvent and the concentration of the reactants. Still another way to form a semipermeable mem-brane is to include a quantity of protein in the temporay cap-sule which can thereafter be crosslinked in surface layers by exposure to a solution of a crosslinking agent such as gluter-aldehyde.
The foregoing process has been used to encapsulate viable islets of Langerhans which, in a medium containing thenutrients and other materials necessary to maintain viability and support in vitro metabolism of the tissue, excrete insu]in in the presence of glucose. Encapsulated tissue has been main-tained in a viable state for three months. Also, liver cells have been encapsulated and have been demonstrated to be in a physiologically active state.
In another aspect, the instant invention provides a tissue implantation method which does not require surgery and

2~ which overcomes many of the problems of immune rejection. In accordance with the invention, the capsules are injected into a suitable site in a mammalian body, and function normally until the tissue expires, or until natural body processes succeed in isolating the capsules so that substances required for viability of the tissueare no longer available~ At this point, because surgery is not required for the implant, fresh tissue may be readily provided by another injection. The mammalian body may accordingly be provided with the specialized function of the tissue as long as desired.
In a preferred embodiment of the invention, mal~lalian ~ ~5~5~

1 islets of Langerhans, or islet preparations containing selected amounts of alpha, beta, and/or delta cells from islets are encapsulated in polylysine and polyethyleneimine cross-linked alginate membranes. These may be periodically injected, e.g., into the peritoneal cavity of a diabetic mammalian body and function as an artificial pancreas.
Accordingly, it is a primary object of the invention to provide a method of encapsulating living cells, organelles, or tissue in a membrane permeable to the nutrients and other ~ substances needed for maintenance and metabolism and to meta-bolic products, but impermeable to bacteria and to substances having a molecular weight above a selected level, so as to e~clude agents responsible for immunological rejection of the foreign tissue. Other objects of the invention include the pl-O-ViSiOIl of encapsulated living tissue useful for producing hor-mones such as insulin and for effecting complex chemical changes cllaracteristic of the in vivo tissue, to provide an insulin generation syst~m~ to provide a body fluid deto~if~in~ s~stemf and to provide encapsulated activated charcoql, Other objects of the invention are to provide a me~hod of implanting living tissue in mammalian bodies and to provide a non-surgical tissue implantation technique. Still another object is to provide a method of encapsulating living tissue which allows the production of capsules having a high surface area to volume ratio and membranes with a preselected in vivo residence time. Another object of the invention is to provide an artificial pancreas.
These and other objects and features of the invention will be apparent from the following description of some preferr-ed embodiments and from the drawing.

5~

_IEF DESCRIPTION OF THE DRAWING
The sole figure of the drawing schematically illustra-tes a preferred method of encapsulating living tissue suitable for use in the process of the invention, and the product micro-capsule.

DESCRIPTION OF THE PREFERRED EMBODIMENT
The tissue, organelle, or cell to be encapsulated is prepared in accordance with well-known prior art techniques in finely divided form and suspended in an aqueous medium suitahle for maintenance and for supporting the ongoing metabolic pro-cesses of the particular tissue involved. Media suitable forthis purpose are available commercially. The average diameter of the material to be encapsulated can vary widely between less than a micron to several millimetersO Mammalian islets of Langerhans are typically 1~0 to 200 microns in diameter. Of course, individual cells such as pancreatic beta cells, alpha cells, delta cells, or various ratios thereof, whole islets of Langerhans, individual hepatocytes, organelles, or other tissue units may be encapsulated as desired. Also, microorganisms may be encapsulated as well as non-living materials such as ~iolog-ical materials.
The ongoing viability of such living matter is depen-dent, inter alia, on the availability of required nutrients, oxygen trans~er, absence of toxic substances in the medium and the pH of the medium. Heretofore, it has not been possible to maintain such living matter in a physiologically compatible environment while simultaneously encapsulating. The problem has been that the conditions required for membrane formation have been lethal or harmful to the tissue, and no method of membrarle formation which tissue can survive in a healthy state has been ~5;~

forthcoming. It has now been discovered that certain water sol-uble substances which are physiologically compatible with living tissue and can be rendered water insoluble to form a shape-retaining, coherent mass can be used to form a "temporary cap-sule" or protective barrier layer ahout tissue particles. Such a material is added, typically at low concentration, to the tissue culture medium. The solution is then formed into drop-lets containing tissue together with its maintenance medium and is immediately rendered water insoluble and gelled~ at least in a surface layer. Thereafter, the shape-retaining temporary capsules are provided with a permanent semipermeable membrane.
Where the material used to form the temporary capsules permits, the capsule interior may be reliquified after formation of the permanent me1-nbrane. This is done by re-establishing the con-ditions in the medium at which the material is soluble.
The material used to form the temporary capsules may be any non-toxic, water-soluble mate:rial which, by a change in the surroundlng temperature, p~I, or ionic environment or con-centration, can be converted to a shape retaining mass. Pre-ferably, the material also contains plural, easily ionized groups, e.g., carboxyl or amino groups, which can react by salt formation with polymers containing plural groups which ionize to form species of opposite charge. As will be explained below, this type of material enables the depositon of a permanent mem-brane of a selected porosity and a selected in vivo lifespan in surface layers of the temporary capsule.
The presently preferred materials for forming the tem-porary capsule are water-soluble, natural or synthetic poly-saccharide gums. Many such materials are commercially available.
They are typically extracted from vegetable matter and are of-ten 2~3 1 used as additives to various foods. Sodium alginate is the pre-sently preferred water soluble gum. Other useable gums include guar gum, gum arabic, carrageenan, pectin, tragacanth gum, xanthan gum, or their acidic fractions.
These materials comprise glycoside-linked saccharide chains. Many contain free acid groups, which are often present in the alkali metal ion form, e.g., sodium form. If a multi-valent ion such as calcium or strontium is exchanged for the alkali metal ion, the liquid, water-soluble polysaccharide mole-1~ cules are "crosslinked" to form a water insoluble, shape-retain-ing gel which can beresolublized on removal of the ions by ion exchange or via a sequestering agent. While essentially any multivalent ion which can form a salt is operable, it is pre-ferred that physiologically compatible ions, e.g., calcium, be employed. This tends to pr~serve the tissue ln the living state.
Other multivalent cations, can be used for less fragile material.
Other gums 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-gum solution composition comprises equal volumes of tissue in its medium and a one to two percent solution of gum in physiological saline. When employing sodium alginate, a 1.0 to 1.5 percent solution has been used with success.
When encapsulating materials which can resist chanses in temperature~gelatin or agar may be used to form the temporary capsuies. These can be gelled by injection into a low temper-ature environment. Other water soluble substances such as hydr-oxyethyl methacrylate may also be used.
In the next step o~ the encapsulation process, the gum S~51~

1 solution containing the tissue is formed into droplets of a desired size. Thereafter, the droplets are immediately gelled to form shape-retaining spherical or spheroidal masses. Apparatus for conducting these latter steps is illustrated at s-tep BC of the drawing. A beaker 10 containing an aqueous solution of multivalent cation, e.g., 1.5 percent CaC12 solution~ is fitted with a magnetic stirring bar 11 and stirrer 12. The stirring mechanism is actuated to produce a vortex 14 having a hollow center 16. A capillary tube 18 of a selected inside diameter is positioned within hollow region 16 of the vortex and fitted with a vibrator 20. The supension containing tissue and the solubi~
lized gum is fed through the capillary. The effect of surface tension which would induce the formation of relatively large droplets is minimized by the vibrator so that droplets, illustr-ated at 22, of a size comparable to t:he inside diameter of the capillary, are shaken of~ of the capillary tip. These inmediate]y contact the solution where they absorb calcium i~ms. This results in "crosslinking" of tlle gel and in the formation of a shape-retaining, high viscosity prot~ctive temporary capsule XO containing the suspended tissue and its medium. The capsules collect in the solution as a separate phase and are separated hy aspiration.
In an alternative embodiment of the process, a small amount of polymer of the type used for permanently crosslinking the gum is included in the solution together with the multivalent ions (or other solution capable of gelling the particular gum employed). This results in the formation of permanent crosslinks.
Capsules of this type have certain advantages if the goal is to preserve the tissue.
In the next step of the process, a semipermeable mem-1 brane is deposited about the surface of the temporary capsules.
There are a variety of methods available for effecting this step, some of which are known in the art. For example, inter-facial polymerization techniques can be exploited. In inter-facial polymerization, a pair of at least difunctional mutually reactive monomers, or a monomer and a relatively low molecular w~ight polymer, one of which is soluble in polar solvent such as water and the other of which is soluble in hydrophobic sol-vents such as hexane, are caused to react at the interface of an ~ emulsion of the water-in-oil type. In accordance with the procedure disclosed in the Lim et al application noted above, the material to be encapsulated is suspended or dissolved in water together with the water soluble component of the reac~ion, the aqueous phase is emulsified in a hydrophobic solvent, and the complementary monomer is added to the continuous phase of the system so that polymerization occurs about the aqueous drop-lets. By controlling the nature of the continuous phase solvent and the concentration of the reactant contained therein, it is possible to exercise control over pore size and to produce semi-permeable microcapsules.

This technique may be used in accordance with the instant invention if the water soluble reactant 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 a hexane-chloroform mix. The com-plementary monomer is next added, preferably incrementally, to induce interfacial polymeri2ation at the surface of the aqueous droplets. Because of the gelled mass of polysaccharide sur-rounding the suspended tissue, and especially if suitably bu--fered polyfunctional amino-group containing polymers such as cer--S~5~

1 tain proteins are employed as the water-soluble reactant, the process is such that the tissue survives the encapsulation in a healthy condition. The substances useful in forming membranes with the polyfunctional amines includes diacids, diacid halides, and multifunctional sulfonyl halides. In addition to the poly-amines, diamines, polyols, and diols may be used. Molecules containing plural amine groups may also be crosslinked with glu-taraldehyde to form a membrane. Another useful method of mem-brane formation is by interfacial polvmerization utilizing poly-~ addition reactions. In this case, for example, multifunctionalamines absorbed in surface layers of the temporary capsules are reacted with epichlorohydrin, epoxidized polyesters, or diisocy-anate.
The preferred method of forming the membrane, illustr--ated as step D in the drawing, is to permanently cross link surEace layers of the droplets by subjecting them to an a~ueous solution of a polymer containing groups reactive with function-alities in the gel molecules. Certain long chain quaternary ammoniu~ salts may be used for this purpose in some circumstances.
When acidic gums are used, polymers containing acid reactive groups such as polyethyleneimineand polylysine may be used. In this situation, the polysaccharides are crosslinked by inter-action between the carboxyl groups and the amine groups.
Advantageously, permeability can be controlled by selecting the molecular weight of the crosslinking polymer used. For example, a solution of polymer having a low molecular weight, in a given time period, will penetrate further into the temporary capsules than will a high molecular weight polymer~ The degree of penet~a~

tion of the crosslinker has been correlated with the resulting permeability. In ge~era1, t`~ higher the molec~lar weight aDd 5~8 the less penetration, the larger ~le pore size. Broadly, poly-mers within the molecular weight range of 3,000 to lOO,OOO
daltons or greater may be used, depending on the duration of the reaction, the concentration of the polymer solution, and the de~ree of permeability desired. One successful set of reaction conditions, using polylysine of average molecular weight of about 35,000 daltons, involved reaction for two minutes, with stirring, of a physiological saline solution containing 0.0167 percent polylysine. Optimal reaction conditions suitable for controlling permeability in a given system can readily be deter-mined empirically without the exercise of invention.
The selection of the cross-linker~s) also determines the in vivo residence time of the capsules. In the system described above, the permanent capsule membrane comprises poly-saccharide (a readily injestible substance) cross-linked with either or both a polypeptide or protein, e.g., polylysi.ne, or a synthetic substance, e.g., polyethyleneimine. Polymers vary with respect to the rate at which they can be dispersed in vivo.

Some are digested without difficulty, e.g., protein; others are slowly degraded, and still others remain indefinitely. The process of the invention contemplates cross-linking with one or more pol~mers to produce capsules havin~ a selected rate of dis-solution in vivo, ranging generally between a few hours or days to substantial permanence. The example which follows discloses how to produce capsules which remain intact at least about two months within the peritoneal cavity of rats. However, the inven-tion is not limited to these particular capsule membranes nor to capsules of this degree of in vivo life. In fact, the optimal in vivo life of the microcapsules depends upon their extended use and their site of implantation. Those skilled in the art ~ ~S~5~

will be able to produce microcapsules of a selected in vivo life-span empirically without the exercise of invention in view o~
this disclosure.
At this point in the encapsulation, capsules may be collected which comprise a permanent semipermeable membrane sur-rounding a gelled solution of gum, tissue compatible culture medium, and tissue particles. If the object is simply to pre-serve the tissue in a protective environment, no further steps need be done. However, if mass transfer is to be promoted with-in the capsules and across the membranes, it is preferred to re~

liquify the gel to its water soluble form. This may be done by reestablishing the conditions under which the gum is a liquid, e.g., changing the pII of the medium or removing the calcium or other multifunctional cations used. III the gels which are illSO-luble in the presence of multivalent cations, the medium in the capsule can be resolubilized simply by immersing the capsules in phosphate buffered saline, which contains alkali metal ions and hydrogen ions. Monovalent ions exchange with the calcium or other multifunctional ions within the gum when, as shown at stage E of the drawing, the capsules are immersed in the solution with stirring. Other salts, e.g. sodium citrate, may be used for the same purpose.
Iastly, depending on the type of semipermeable membrane formation technique employed, it may be desirable to treat the capsules so as to tie up free amino groups of the ~ike which would otherwise impart to the capsules a tendency to clump. This can be done, for e~ample, by immersing the capsules in a solutio~
of sodium alginate.

The invention contemplates the injection of encapsul-ated, finely divided tissue, multicellular fractions thereof, or ~ ~ ~5;~

1 individual cells into an appropriate site ~ithin a mammalian body for the purpose of providing the body, at least temporarily, with the tissue's specialized physiological function. The pro-cedure has the dual advantages of obviating the need for sur-gical implantation (although capsules may be implanted surgi-cally if desired) and successfully dealing with the problems of immune rejection and natural physical isolation. Preferably, the capsule membranes consist of substances which are injested after expiration of the tissue. As noted above, this can be accomplished by employing a cross-linker whichresists in vivo breakdown so that a given useful in vivo life is attained.
From the foregoing it will be apparent that the encap-sulation process and implantation technique of the invention can be practised using a wide variety of reagents and encapsulated materials and can be varied significantly without departing from the scope and spirit of the invention. The following example should accordingly be construed in all respects as illustrative and not in a limiting sense.

Example Islets o~ ~angerhans were obtained from rat pancreas and added to a complete tissue culture (CMRL-1969 Connaught Laboratories, Toronto, Canada) at a concentration of approxima-tely 10 islets per milliliter. The tissue culture contains all nutrients needed for continued viability of the islets as well as the amino acids employed by the Beta cells for making insulin. Four-tenths of a milliliter of the islet suspension was 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 were p~aced in a 150 milliliter beaker on a stirrer and ~s~

1 stirred at a rate which induced the formatlon 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 dia~
meter about 300-microns was then fitted with a vibrator ¢60 cycles per second~. The capillary tip was then placed within the center of the vortex, the vibrator turned on, and the sod-ium alginate-culture medium-tissue suspension was forced there-through with an infusion pump~ Droplets on the order of 300 -400 microns in diameter are thrown from the tip of capillary and immediately enter the calcium solution.

After 10 minutes, the stirrer was turned off and thesupernatant solution was removed by aspiration. The gelled cap-sules were then transferred to a beaker containing 15 ml of a solution comprising one part of a 2~ 2 (cyclohexylamino) ethane sulfonic acid solution in 0.6% NaCl (isotonic, pH=~.2) diluted with 20 parts 1% CaC12. After a 3 minute immersion, the cap-sules were washed twice in 1% CaCl~.
The capsules were then trans~erred to a 32 ml solution comprisin~ 1/80 o~ one percent polylysine (a~erage ~lW 35,000 A~iU) in physiological saline. After 3 minutes, the polylysine solu-tion was decanted. The capsules were then washed with 1% CaC12, and then suspended for 3 minutes in a solution of polyethylene-imine ~MW 40,000 - 60,000) produced by diluting a stock 3.3%
polyethyleneimine solution in morpholino propane sulfonic acid buffer (0.2M, pH=6) with sufficient 1% CaC12 to result in a final polymer concentration of 0~12%. The resulting capsules, having permanent semipermeable membranes, are then washed twice with 1% CaC12, 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 5~

1 contain lslets o~ ~angerhans. Gel on the interior of the cap-sules 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.
Under the microscope, these capsules have an appearance illustrated in the drawing. They comprise a very thin membrane 24 which encircle an islet 26 within which individual cells 28 can be seen. Molecules having a molecular weight up to about 100 thousand can traverse membrane 24. This allows oxygen, amino ~ acids, nutrients, and plasma components used in culture media (e.gO, fetal calf plasma components) to reach the islet and allows insulin to be excreted.
Example 2 ~ fter repeated washings in physiological saline, micro-capsules made in accordance with Example 1 containing approxima-tely 15 islets were suspended in 3 milliliters of CMRL-1969.
When eight days old, in the presence of 600 mg/dl glucose, the capsules ~xcreted, in one run, 67 unit:s/ml insulin in 1.5 hours.

In a second run, 6~ units/ml insulin were produced ln the same amount of time. One ~eek old capsules, in the same medium, but in the presence of 100 mg/dl glucose, in a first run, excreted 25 units/ml insulin in 1.2 llours, and in a second run, excreted 10 umits/ml.
Example 3 Diabetic rats with blood glucose levels in the range of 500-700 mg/dl were each treated with approximately 103 islets encapsulated as set forth in Example 1, and suspended in phy-siological saline. The capsules were introduced by injection into the peritoneal cavity using a number 19 needle fitted to a syringe. ~lood sugar levels were assayed daily and uniformly 1 ~ound to be below 300 mg/dl. Animals sacrificed after two months showed no signs of to~ic reaction about the site of the implantation Capsules removed from sacrificed animals after a two-month in vivo life were intact and showed no signs of de-gredation.
EXAMPLE 4 : ENCAPS~LATION 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 in place of the 0.4 ml islet suspension. The ongoing viability of the liver tissue has been demonstrated by the dye exclusion technique (trypan blue exclusion). It is known that liver tissue, in vitro, can ingest toxins from its environment.
Accordingly, toxins of a molecular weight low enough to pass through the semipermeable membranes are injested and destroyed by the tissue. Essentially all toxins treated by the liver are low molecular weight materials. However, the toxins may be protein-complexed. Capsular permeability can be varied according to the need.

The procedure of e~ample 1 is repeated except that par-ticulate activated charcoal i5 suspended directly in the sodium alginate, solution, the half milliliter of tissue suspension is omitted, and polylysine of an average molecular weight of 35,0Q0 is used as a crosslin~er. As long as the charcoal partic]es are smaller than the smallest inside diameter of the capillary used to produce the droplets, charcoal of high surface area surrounded by a semipermeable membrane results. These effectively prohibit the escape of charcoal chips or dust, yet can be used to absorb medium range molecular weight materials (up to about 2,000 daltons) from fluid passed about the capsules.

1 The operability of the process has been demonstrated with other living cells including red blood cells, using serum as a medium, sperm cells, using semen as the medium, and baker's yeast. Those skilled in the art will appreciate that a ~ariety of other materials can be encapsulated in addition to those specifically set forth herein, and that permeability can be controlled as desired for selected applications of the process.
Accordingly, other embodiments are within the following claims.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for encapsulating viable tissue within a semipermeable membrane, said process comprising the steps of:
A. suspending finely divided living tissue in an aqueous medium which is physiologically compatible with the tissue and which contains a water soluble substance which (a) is physiologically compatible with the tissue; and (b) can be reversible gelled to form a coherent, shape-retaining mass;
B. forming the suspension into droplets of a size sufficient to envelop tissue;
C. gelling the droplets to form discrete, shape-retaining temporary capsules; and D. forming a permanent semipermeable membrane about the temporary capsules.

2. A process for encapsulating a core material within a semipermeable membrane, said process comprising the steps of:
A. placing the material in a solution of a water-soluble substance that can be reversibly gelled;

B. forming the solution into droplets;
C. gelling the droplets to produce discrete shape-retaining temporary capsules; and D. forming semipermeable membranes about the temporary capsules.

3. A process as claimed in claims 1 or 2 further including the step of reliquifying the gel within said membranes.

4. A process as claimed in claim 2 wherein said substance comprises a gum.

5. A process as claimed in claim 4 wherein said gum has free acid groups and said membrane formation step is effected by contacting the temporary capsules with a polymer of a molecular weight between 3000 and 100,000 daltons and having free amino groups, said contacting being effective to form permanent polymer crosslinks between acid groups in a surface layer of the capsule.

6. A process as claimed in claim 5 wherein the polymer used for crosslinking is selected from the group consisting of polylysine and polyethylenimine, said polymer having an average molecular weight of about 35,000 daltons.

7. A process as claimed in claim 2 wherein the membrane is formed by an interfacial polymerization wherein the temporary capsules are used as a core material in the aqueous phase of a water-in-oil emulsion.

8. A process as claimed in claim 7 wherein the reactants used in the polymerization are selected from the group con-sisting of water soluble polyols, diols, polyamines, and diamines, and water immiscible diacid halides, diacids, and multifunctional sulfonyl halides.

9.0 A process as claimed in claim 7 wherein the interfacial polymerization is a polyaddition reaction.

10. A process as claimed in claim 2 wherein the material is selected from the group consisting of enzymes, immunoproteins, activated charcoal particles, and viable tissue.

11. A process as claimed in claim 4 wherein the gum is an alkali metal alginate.

12. A process as claimed in claim 4 wherein the gum comprises a polysaccharide containing free acid groups.

13. A process for encapsulating a core material within a semipermeable membrane, said process comprising the steps of:
A. suspending the core material in an aqueous medium which contains a water-soluble gum containing acid groups;
B. forming the suspension into droplets;
C. subjecting the droplets to a solution of multi-valent, cations to gel the droplets as discrete, shape-retaining, water insoluble temporary capsules; and D. permanently cross-linking surface layers of said temporary capsules to produce a semipermeable membrane about said droplets by subjecting them to a polymer containing substituents reactive with the acid groups of said gum.

14. A process as claimed in claim 13 comprising the additional step of resolubilizing the gel within said capsules.

15. A process as claimed in claim 13 wherein the water soluble gum is sodium alginate and the multivalent cation solution is a calcium solution.

16. A process as claimed in claim 15 including the ad-ditional step of removing the calcium ions contained within

Claim 16 continued said capsules to resolubilize the gelled alginate interior of the membranes of said capsules.

17. A process as claimed in claim 15 wherein said core material is a mammalian tissue selected from the group consist-ing of Islets of Langerhans, liver, and individual cells thereof and said medium is a physiologically compatible tissue medium.

18. A process as claimed in claim 13 wherein said core material is a mammalian tissue selected from the group con-sisting of Islets of Langerhans, liver, and individual cells thereof and said medium is a physiologically compatible tissue medium.

19. A process as claimed in claim 13 wherein said core material comprises living tissue and said aqueous medium is a complete tissue culture medium sufficient to maintain said tissue in vitro.

20. A process as claimed in claim 13 wherein said cross-linking polymer is selected from the group consisting of polylysine and polyethylenimine.

21. A process as claimed in claim 20 wherein the average molecular weight of said polymer is about 35,000 daltons.

22. A process as claimed in claim 13 wherein said forming step is effected by forcing droplets of the suspension through a capillary tube while vibrating the tube within the center of a vortex formed by imparting circular fluid motion to an aqueous solution containing multi-valent cations.

23. A process as claimed in claim 2 wherein said core material is selected from the group consisting of hormones, enzymes and antibodies.

24. A process as claimed in claims 1, 2 or 13 wherein said core material is selected from the group consisting of biological materials and living cells.

25. Viable tissue encapsulated within a semipermeable membrane prepared by a process as claimed in claim 1, 2 or 13, or an obvious chemical equivalent thereof.

26. A core material encapsulated within a semipermeable membrane prepared by a process as claimed in claims 1, 2 or 13, or an obvious chemical equivalent thereof.

27. Viable tissue encapsulated within a semipermeable membrane wherein said viable tissue comprises a mammalian islet of Langerhans or a fraction thereof, whenever prepared by a process as claimed in claims 17 or 18 or an obvious chemical equivalent thereof.

28. Viable tissue encapsulated within a semipermeable membrane wherein said viable tissue comprises a mammalian islet of Langerhans or a fraction thereof, thereof maintaining culture medium whenever prepared by a process as claimed in claim 19 or an obvious chemical equivalent thereof.

29. Viable tissue encapsulated within a semipermeable membrane wherein said viable tissue comprises mammalian liver tissue or a fraction thereof whenever prepared by a process as claimed in claims 17 or 18 or an obvious chemical equiv-alent thereof.

30. Viable tissue encapsulated within a semipermeable membrane wherein said viable tissue comprises one or more pancreatic endocrine cells whenever prepared by a process as claimed in claim 23 or an obvious chemical equivalent thereof.