CA1184518A - Reversible microencapsulation - Google Patents
Reversible microencapsulationInfo
- Publication number
- CA1184518A CA1184518A CA000398210A CA398210A CA1184518A CA 1184518 A CA1184518 A CA 1184518A CA 000398210 A CA000398210 A CA 000398210A CA 398210 A CA398210 A CA 398210A CA 1184518 A CA1184518 A CA 1184518A
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- polymer
- membrane
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- stripping
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5073—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/20—After-treatment of capsule walls, e.g. hardening
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0012—Cell encapsulation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K2035/126—Immunoprotecting barriers, e.g. jackets, diffusion chambers
- A61K2035/128—Immunoprotecting barriers, e.g. jackets, diffusion chambers capsules, e.g. microcapsules
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/74—Alginate
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Dispersion Chemistry (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biomedical Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Medicinal Preparation (AREA)
- Manufacturing Of Micro-Capsules (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
Abstract
Abstract of the Disclosure Disclosed is a process for microencapsulating a core material and subsequently releasing the core material by selectively disrupting the membranes of the microcapsules. The encapsulation technique involves the formation of a semipermeable membrane, e.g., around a droplet, through the formation of multiple ionic salt bonds between a polyionic polymer in the droplet and a crosslinking polyionic polymer which possesses multiple ionic groups of opposite charge. The membrane can be selectively disrupted by exposing it first to a solution of com-peting crosslinking multivalent (preferably di or trivalent) ions followed by a solution of a competing polyionic polymer of the same charge as the polymer in the original droplet.
Alternatively, a mixed solution of the two competing solutions may be used together. For example, a membrane comprising anionic alginate salt bonded to cationic protein can be selectively disrupted by exposing the membrane to a mixed solution of mona-tomc, multivalent cations, e.g. Ca++ ions, and a water-scluble polymer have plural anionic moieties, e.g., heparin, and sub-sequently sequestering the monatomic cations.
The process may be used to encapsulate and subsequently release cell cultures without damage to the cells.
Alternatively, a mixed solution of the two competing solutions may be used together. For example, a membrane comprising anionic alginate salt bonded to cationic protein can be selectively disrupted by exposing the membrane to a mixed solution of mona-tomc, multivalent cations, e.g. Ca++ ions, and a water-scluble polymer have plural anionic moieties, e.g., heparin, and sub-sequently sequestering the monatomic cations.
The process may be used to encapsulate and subsequently release cell cultures without damage to the cells.
Description
~.4q5~L~
BackgroUnd This invention relates to a method of encapsulation which is reversible, that is, a method which may be used to encapsulate a liquid or a solid material and thereafter to release the material by selectively disrupting the capsule membranes~ An important embodimen-t of the invention involves the microencapsulation of living cells which may subsequently be released from within the produced capsule membranes without damage.
Canadian Application Serial No. 348,524, now Patent No. 1,145,258, entitled "Encapsulation of Viable Tissue and Tissue Implantation Mlethod", by F. Lim discloses a micro-encapsulation technique which can be used to encapsulate esscntially any solid or liquid material within semipermeable or substant:ially impermeable capsule membranes. An outstan~ing advantage of the process is that the conditions under which th~ capsule membranes are formed involve no toxic or denatur-.ing reagents, ~xtremes of temperature, or other conditions which damacJe living cells. The process of that application is ~ according:ly well-suited for the production of microencapsulated l:Lving materials which remain viable and in a healthy state.
Because the process allows a degree of control of the perme-~bility o~ the membrane, it is now possible to microencapsulate cell cultures of procaryotic, eukaryotic~ or other origin such that cells of the culture are protected from contaminating bacteria, high molecular weight immunoglobulins, and other potentially deleterious factors, and remain confined within a microenvironment well-suited for their continuing viability and ongoing metabolic functions. If the ~sj D~
1 microcapsules are suspended in a conventional culture medium suf-ficient to suppo~t growth of the living cells involved, the microencapsulated cells are free ~o ingest substances needed for metabolism which diffuse through the membrane and to excrete their me~abolic products through the capsule membrane into the surrounding medium.
5~
1 Summar~ of the Invention The instant invention is directed to a method of selec-tively disrupting certain membranes synthesized during the micro-encapsulation procedure se~ forth in the above-referenced application without any detectable damage to the encapsulated core rnaterial, ~ore specifically, the process of this invention is practiced on membranes comprising a water-insoluble matrix formed froTn at least two water-soluble componen~s: a polyme_ which includes multiple cationic moieties [polycationic polymer, e.g. polye-thylene amine); and a polymer having multiple anionic rnoieties (polyanionic polymer, e.g. sodium alginate gum). The two components are connected by ~alt bridges between the anionic and cationic moieties to form the matrix.
The process of the invention comprises the steps of expo~ing membranes of the type set forth above to a solution of ca~ions, peferably, monatomic or very low molecular weight multi-~alent cations/ and a solution of a stripping polymer having plural anionic rnoieties. Preferably, these solutions are mixed together. The anionic charge of the polymer ~hould be sufficient to disrupt the salt bridges and should preEerably be equal to or yreater than the charge density of the polyanionic polymer of the membrane. The solution is contacted with the capsules to allow the cations, e.g., calcium or aluminum, to eornpete against the polycationic polymer in the capsule m~m~rane for anionic sites on the polyanionic polymer. Sirnul~aneously, the stripping polymer having plural anionic moieties competes with the polyanionic polymer for cationic sites on the polymer chains~ This results ; in "softening" or "unæipping" of the capsule membranes. To 1 complete the disruption, the capsules are wa8hed a~d then exposed to a sequestering agent to r~move cations associated with the polyanionic polymer~ The preferred sequestering agents are che-lating ayents such as citrate ion~ or EDTA ions. If~ as in an important embodiment, the capsules con~ain viable cells, it is preferred to mix the sequestering agent with an isotonic saline solution.
The currently preferred cation is calciumO Examples of the stripping polymer having plural anionic yroups used in the mixed solution include polysulfonic acids or (pre~erably) their salt~, either natural or synthetic. Outstanding results have been obtained using heparin, a natural polymer containing plural ~ulfonate groups. Polymers containing polyphosphoric or polyacrylic acid salt moie~ies may also be used. The currently preferred seques~ering agent is sodium citrate.
The invention also contemplates a method of encap-sulati.ng viabLe cells within a pro~ective environment and sub-sequently releasing the cells. Thus, the invention provides what may be described as a package which maintains living cell cultures of whatever origin in a sterile, stablizing environment in which they can undergo normal metabolism and even mitosis and from which they subsequently can be released.
Accordingly, an object of the invention is to provide a method for encapsulating living cells within permeable membranes and subsequently selectively disrupting the membranes to release the cells. Another object is to provide a process for selec-tively disrupting membranes. Another object is to disrupt mem-1 branes without damage to proximate living tissue. Still anotherobject is to provide a method of encapsulatiny and subsequently releasing finely divided materials J liquids J and solutions.
These and other objects and features of the invention will be apparent from the following description of some important embodirnents.
Description The selective membrane disruption process of the inven-tion is practiced on membranes consisting of a salt bridge-bonded ]0 matrix of a polycationic polymer and a polyanionic polymer.
U~ually, the membranes will have a spheroidal form defining an enclo~ed int~rior containing an encap~ulated substanceO However, ~he proce~s may al50 be practiced on membranes o~ this type which take other than spheroidal form, Although es~entially any material ~compatible with aqueous environments) in liquid or solid form can be encapsulated and subsequently released without damage by the process of this irlvention, its most notable utility, as prPsently contemplated, lies in it~ abili~y ko encapsulate and subsequently release living systems such as cell cultures. Accordingly, the descrip-tion which follows will be primarily confined to a discussion of the encapsulation and release of cell~. Those skilled in the art will be able to adap~ ~he process withou~ difficulty to the encapsulation of less fragile materials.
lation __.
The tissue or cells to be encapsulated are suspended in an aqueous medium ~uitable for maintenance and for supporting the 1 ongoing me~abolic processes of the particular cell type involved.
Media sui~able for this purpose are well known to those sXilled in the art and often ar~ availahle commercially. The average diameter of the cell mass or ot~er material to be encapsula~ed can vary widely between a few microns to a millimeter or more.
Mammalian Islets of Langerhans, for examples, are typically 50 to 200 rnicrons in diameter. Tissue fragments and individual cells such as fibroblasts, leukocytes, lymphoblastoids, pancreatic beta, alpha or delta cells, islet of Langerhans, hepatocytes, or the cells of other tisgue may be encapsulated as desired~ Also, microorganisms may be encapsulated including those which have been genetically modified by recombinant DNA or other techniques, The ongoing viability of such living matter is depen-dent, inter alia, on the availability o~ required nutrients, oxy-gen transfer, absence of toxic substances in the medium, and the pH o~ the medium. Heretofore, it has not been possible to main-tain suc~l living rnatter in a physiologically compatible environ-ment while simultaneouæly encapsulating, The problem has been that the conditions required for membrane formation have been ~0 l~tllal or harmful to the tifisue, and prior to the invention of the above-referenced application, no method of membrane ormation which allowed tissue ~o survive in a healthy state had been forthcoming.
However, it has been discovered that certain water-~oluble substances which are physiologically compatible with living tissue and can be rendered water-insoluble to form a æhape-retainingO coherent mass, can be used to form a "temporary cap~ule" or protective barrier layer about individual cells, --7~
l groups of cells, or tissues. Such a subs~ance is added, typically at a concen~ration on the order of 1-2 weight percent to the tissue culture medium. The solution is then formed into droplets containing tissue together with its maintenance or growth 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 more permanent membrane which, in accordance with this invention, may be subsequently ~electively disrupted to release the encapsulated tissue without damage. Where the ma~erial used to form the temporary capsules permi~s, the capsule interior may be reliquified ater formation of the permanent membrane. This is done by re-establishing the conditions in the medium at which the ma~erial is soluble.
The material used to form the temporary capsules may be any non-toxic, water-soluble material which, by a change in ~he ~urrounding io~ic environment or concentration, can be converted to a shape-retaining mass. The material also comprises plural, easily ionized anionic moieties, e.g., carboxyl grc,ups, which can react by sal~-bond forrnation with polymers containing plural cationic groups. As will be explained below, this type of material enables the d~position of the permanent membrane of a selected permeability (including substantially non-porous to a level of several hundred thousand daltons).
The presently preferred polyanionic material for forming the temporary capsule ars acidic, water-soluble, natural or syIl~hetic polysaccharide gumsO Many such materials are com-mercially available~ They are typically extracted from vegetable 1 matter and are often used as additives ~o various foodsO Sodium alginate i3 the presently preferred anionic polymer. Alginate in the molecular weigh~ range of 150,000~ daltong may be used, but because of its molecular dimensions will usually be unable to permeate the finally formed capsule mernbranes. To make capsules without trapped liquid alginate, lower molecular weight alginate, e.g., 40,000--80,000 daltons can be used. In the inished cap-sule, the alginate can then be more easily removed from the intracapsular volume by diffusion through a membrane of suf-ficient porosity. Other useable polyanionic yums include acidic fractions of guar gum, carageenan, pectin, tragacanth gum, or x~nthan gurQ.
These materials compriee glycoside-linked saccharide chain~. Their free acid groups are often present in the alkali m~tal ion form, e.g., ~odium form. If a multivalent ion such as calcium, strontium, or aluminum is exchanged for the alkali metal ion, the liquid, water-soluble polysaccharide rnolecules are "croas-linked" to forrn a water-in~oluble, shape retaining gel which can be resolublized on rernoval of the ions by ion exchange or via a suquestering agent. While essentially any multivalent ion which can form a salt with the acidic gum is operable, it is preferred that physiologically compatible ions, e.g., calcium, be employed. This tends to preserve the tissue in the living state.
Other multivalent cations can be used for less fragile materialO
Magrlesiurn ions are ineffective in gelling sodium alginate.
A typical solution composition comprises equal volumes o a cell suspensiorl in its medium and a one to two percent solu-tion of gum in physiological saline. When employing sodium algi-nate, a 1.2 to 1.6 percent solution has been used with success.
1 In the next s~ep of the encapsulation process, the gum solution containing the tissue i5 formed into droplets of a desired size sufficient to envelop the cells ~o be encapsulated.
Thereafter, the droplets are immediately gelled to form shape-re~aining spherical or spheroidal masges~ The drop formation may be conducted by known techniques.
A tube containing an aqueous solution of rnultivalent cations, e.g., 1.5~ CaC12 solution, is fitted with a stopper which holds a drop forming apparatus. The appartus comprises a housing having an upper air intake nozzle and an elongate hollow body friction fitted into the stopper. ~ lO cc syringe equipped with a stepping pump is mounted atop the housing with, e~g., a O.Ol inch I.D. Teflon coated needle passing through the length of the housing. The interior of the housing is designed such that the tip of the needle is subjec~ed to a constant laminar air flow which acts as an air knife. In use, with the syringe full of ~olution cont~ining the rnaterial to be encapsulated, the stepping pump i~ actuated to incrementally force droplets of solution from the tip o~ the needle~ Each drop is "cut of-f" by the air stream and fall~ approximately 2.5 cm into the CaC12 solution where it is immedia~ely gelled by absorption of calcium ions. The distance between the tip of the needle and the surface of the CaC12 solution is great enough, in this instance, to allow the sodium algina~e/cell suspension to assume the most physically favorable æhape; a æphere (maximurn volume for minimwn surface area). Air within the tube bleeds through an opening in ~he stopper. This results in "cross-linking" of the gel and in the formation of a high viscosity, shape-retaining protective tem-1 porary capsule containing the suspended ti~sue and its medium.The capsules collect in the solution as a separate phase and are separated by aspiration.
In the next step of the proc~ss, a membrane is deposited about the eurface of the temporary capsules by cxoss-linkiny surface layers. This is done by subjecting the temporary cap~ules comprising polyanion to an aqueous solution of a polymer containing cationic groups reactive with anionic functiorlalities in the polyanionic polymer. Polymers containing reactive cationic groups such as free amine groups or combina~ions of amine and imine groups are preferred. In this situation/ the polysaccharide gurn is crosslinked by interaction (sal~ bond ~ormati.on) between the carboxyl groups and the amine or imine groups of the polycationic polymer. Advantageously, permeability can be controlled within limi~s by selecting the molecular weight o~ the cro~s-linking polymer used and by varying exposure time and the concentration of poLymer in solution. A solution of polymer having a low molecular weight, in a given time period, will penetra~e further into the temporary capsules than will a high molecul~r weight polymer. The degree of penetration of the cross-linker has been correlated with the resulting permeability.
In general, the higher the molecular weight and the less penetra-tion, the larger ~he pore size. Longer exposures and more con-centrated polymer solutions tend to decrease the resulting mernbrane'~ upper limit of permeability However, the average molecular weight of the polymer is the dominarlt determinant.
Broadl~, polymers within the molecular weight range o~ 10,000 to 100,000 daltons or greater may be used, depending on the duratio 1 of the reaction, th~ concentration of the polymer solution, and the degree of permeability desirzd. One successful ~et of reac-tion conditions, using polylysine of average molecular weight of about 35,000 daltons, involved reaction for three minutest with stirring, o a physiological saline solution containing 0~0167 percent polylysine~ This results in membranes having an upper limit of permeability of about lOOrOOO daltons. Generally, higher molecular weight materials form membrane which are more difficult to subsequently disrupt as compared with lower molecular weight ma~erials. The charge densi~y of the crosslinking polycationic polymer also affects the pore size and ea~e of membrane disruption. Generally, higher charge density materials form less porous membranes which are more di~ficult to dlsrupt. Optimal reac~ion conditions suitable for controlling permeability in a given sys~em can readily be determined emp~rically in view of the foregoing guidelines.
Example~ of suitable cross-linking polymers include proteins and polypeptides, either natural or synthetic, having ~ree amino or cornbinationa of amino and imino groups, polyethyl-enearnines, polyethyleneimines, and polyvinylamines Polylysine,in both the D and L forms, has been used with success. Proteins such as polyarginine, polycitrulline, or polyornithine are also operableO Polymers in the higher range of positive charge den-sity (e.g., polyvinylamine) vigorously adhere to the anionic groups of the polyanionic molecules and are rnore difficult to disrupt.
At this point in the encapsu]ation, capsules may be collected w~ich cornprise a "psrmanent" semipermeable membrane 1 surrounding a gelled solution of gum, cell type compatible culture medium, and the cells. If the obje~t is simply to preserve the cells in a protective environment, no further steps need be done. However, if mass transfer is to be promoted within the capsules and across the membranes, it i6 preferred to re~lj.quify the gel to its water- oluble form, This may be done by reestahlishing the conditions under which the gum is a liquid, e.g., removing the calcium or other multifunctional ca~ions from the gel. The medium in the capsule can be resolubilized simply by immersin~ the capsules in phosphate buffered saline, which contains alkali metal ions and hydrogen ions. Monovalent ions exchange with the calcium or other multifunc~ional ions within the gum when the capsules are immersed in the solution with stirring. Sodiurn citra~e solu~ions may be used for the same purpose, and serve to sequester the divalent ions~ Gum molecules having a molecular weight below the upper limit of permeability o~ th~ rnernbranes may subsequently be removed from the intracap-sular volurne by diffusion.
La3tly, it may be desirable to -treat the capsules so as to tie up free amino groups of the like which might otherwise impart to the cap~ules a tendency to clump~ This can be done, for example, by immersing ~he capsules in a dilute solution of sodium alginate.
From the foregoing it will be apparent that no harsh reagents, extremes of tempera~ure, or other conditions deleterious to the health and viability of the cells need be used in the membrane formation process. Thuss even very sensitive cells such as mammalian hepatocytes, leukocytes, ~ibroblasts, 1 lymphoblasts, and cells from various endocrine ~issues may be encapsulated withou~ difficul~y~ Of course, ce~ls of microbial oriyin such as yeasts, molds, and bacteria which are bet~er adapted to survive in hostle environtnentsl as well as inert reagents, solids, or biologically active materials may also be encapsulated without damage.
Encapsulated cells of the type described above may be suspended in maintenance medium or growth medium for storage or culture and will remain free of bacterial infection. If suspended in growth medium, cells which undergo rni~osis ln vitro will do so within the capsules Normal in vitro metabolism con-tinues provided the faetors needed for metabolic processes are of ~u~iciently low molecular weight that ~hey can penetrate the capsule mellbrane, or are encapsulated together with the cells.
Metaboli.c products of the cells (if of a molecular weight below the upper limit of permeability) penetrate the membrane and ~ollect in the medium. The cells in encapsulated form may be stored, shipped, or cultured as desired, and may be released from their protective environmen~ without damage by means of the following process of selectively disrupting the membranes.
Disruption oi Membrane In accordance with the invention the encapsulated material may be released by a two step process involving commer-cially available reagents having properties which do not adversely affect the encapsulated cells.
Eirst, the capsules are separated from their suspending medium, washed thoroughly to remove any contaminants and then s~
1 dispersed, with agitation, in a separate or preferably mixed solution o~ cations such as calcium ions or ot~er monatomic (low molecular weigh~ multivalen~ cation) and a stripping polymer having plural anionic moieties such as polysulfonic acid groups~
Polymers containing polyphosphoric or polyacrylic acid moieties ma~ also be used. Heparin, a natural sulfonated polysaccharide, is preferred ~or disrupting membranes containing cells. The anionic charge of the stripping polymer used must be sufficient to diRrupt the salt bridges. Thus the anionic charge density may be equal to or preferably greater than the charge density of th~
interior polyanionic polymer (e.g~, ~he gum) originally employed to forrn the membranes. The molecular weight of the stripping polymer should be at least comparable to and preferably greater than the molecular weight of the interior polycationic polymer used in forrning the merabrane. Within the suspension, the calcium ions com~ete with the interior polycationic polymer comprising the membrane for anionic sites on the polyanionic polymer.
5imultaneously, the stripping polymer dissolved in the solution competes wi~h the polyanionic gum in the membrane for cationic ~0 site~ on the polycationic polymer. This results in a water-dispersible or preferPably water-soluble complex of, e.g~, poly-lysine and the polyanionic polymer, and in association of the cations with gel molecules.
This step renders the membrane susceptible to subsequent exposure to a sequestering agent which completes the disruption process by taking up di or trivalent ions from the gel. Typically, capsule membrane debris, if any, which remains in the medium can be separated easily from the cells.
1 The currently preferred ~olution for the first stage of the selective disruption process comprises 1.1~ calcium cloride (w/v) and between 500 to 1,500 units of heparin per milliliter of solu~ion. A volume of microcapsules is added ~o this solution sufficient to constitute between about 20~ and 30~ of the total volume of suspensionO Calcium chloride and heparin are preferred when disrup~ing membranes of cell-containing capsules since both reagents are physiologically compatible with most cells and mini-mize the possibility of cell damage. Mixtures of aluminum salts or other multivalent cations ~but not Mg+~ ions) may also be used together with the polysulfonic or other acid salt of the type set ~orth above.
In general, the concentration of the ions ~nd anionic polymer in the solution used in this step may vary widely.
Optimum concentrations may be readily determined empirically.
The lowe~t operable concentration for a particular batch o~
~ncap~ula~ed cell~ is preferred.
The curren~ly preferred sequestering agent for per-forming the selective disruption is sodium citrate, although other allcali metal citrate salts and alkali metal EDTA may also be used. When sodium citrate is employed, the optimum con~
centration is on the order of 55 mM. Where the cap~ule membranes being disrupted contain viable tissuel it is preferred that the citrate be dissolved in isotonic saline so as to minimize cell darnageO
The inverltion will be further understood from the following non-limiting examples.
1 Capsule Formation Exam~le_l: Encapsulat.ion of Panc eatic Tissue Islets of Langerhans are obtained rom rat pancreas and added to a complete tissue culture (C~RL-1969 Connaught Laboratories, Toronto, Canada) a~ a concentration of approxima-tely 103 i51et3 per rnilliliter, The tissue culture contains all nutrients needed for continued viability of the islets as well as the amino acids employed by the cells for making hormones. Four-tenths of a milliliter of an islet suspension containing approxi-mately 103 islets per milliliter is then added to a one-half milliliter volume of 102 percen~ sodium alginate ~Sigma Chemical Company) in physiological saline.
Next, a 1.5 percent calcium chloride solution is used to gel droplets on the order of 300-400 microns in diameter.
After the supernatant solution is removed by aspiration, the ~lled droplets are transferred to a beaker containing 15 ml o a ~olution cornpri~ing one part of a 2% 2 (cyclohexylamino) ethane sulfonic acid buffer solution in 0~6~ NaCl (isotonic, pH-8,2) diluted with 20 parts 1% CaCl~. After a 3 minute immersion, the capsules are washed twice in 1% CaC12.
The capsules are then transferred to a 32 ml solution comprising 1/80 of one percent polylysine (average MW 35,000 amu~
in physiological saline. After 3 minutes~ the polylysine solu~
tion is decantedO The capsules are washed with 1% CaC12, and optionally re~uspended for 3 minutes in a solution of polyethyl-eneimine (MW 40,000-60,000) produced by diluting a stock 303~
polyethyleneimine solution in morpholino propane sulfonic acid bufer (0.2M, pH=6) with sufficient 1% CaCl~ to result in a final 1 polymer concentration of 0 D 12~. The re~u~ting capsules, having "permanent" semipermeablP membranes, are then washed twice with 1% CaCl ~, twice with physiological saline/ and mixed with 10 ml of 0.12 percent alginic acid solution.
The capsules resist clumping, and many can be seen to contain islet~ of Langerhans. Gel on the in~erior of the cap-sules is reliquified by immersing th~ capsules in a mixture of ~aline and citrate buffer (pH-7~4) for 5 minutes~ Lastly, the capsules are suspended in CMLR-69 medium.
Under the microscope, these capsules are observed to compri e a thin membrane w~ich encircles an islet within which individual cells can be seen. Molecules having a molecular weight up to about one-hundred thousand can traverse the membra-nes. This allows oxygen, amino acids, nutrients, and plasma com-pon~nts used in cul~ure media (e.g,, lower molecular weight fetal cal~ serurn co~nponents) ko reach khe islek and allows insulin to be excreted.
Exam~le 2. _Encapsulation of Hepatocyte~
The procedure of example 1 is repea~ed except ~ha~ 0.5 ml of a liver cell suspension containing about 105 cells per milliliter is used in place of the 0.4 ml islet suspension. The ongoing viability of the liver cells has been demonstrated by the dye exclusion technique (~rypan blue exclusion) and by their observed ability to con~inuously produce urea. It is known that liver tissue, in vitro, can ingest toxins from its environmen~.
Accordingly, toxins of a molecular weight low enough to pass t~rough khe semipermeable membranes are detoxiied by the cells.
1 Example 3_ Activated Charcoal Encaps la~ion The procedure of exarnple l is repeated except that par-ticulate activated charcoal is suspende~ directly in the sodium alginate solution, the half milliliter of tissue suspension is omitted, and polylysine of of an average rnolecular weight of 35,000 is used as a cross-linkerG As long as the charcoal par-ticles are smaller than the smallest inside diameter of the capi.llary used to produce the dropl~ts, charcoal of high surface area surrounded by a semipermeable membrane resultsO These effectively prohibit the escape of charcoal chips or dust, yet can be used to absorb materials of any pre-selec~ed molecular weight ranye from fluid passed through the capsules~
4: Enca~sulation of Human Fibroblasts Human fibroblasts obtained by treating human foreskin tiRsue with trypsin and EDTA for 5 minutes at 37C in a known manner are suspended in a complete growth medium (CML,R 1969, C~nnaught Laboratories) ~upplemented with 40% (v/v) puriEied fetal calf ~erurn, 0.8% sodium alginate (Sigma) and 200 mg/ml puri1ed cal~ skin collagen. The density of the cell suspension is about 1.5 x 107 cells/ml. Temporary alginate capsules are formed as set forth abovel Semipermeable membranes are deposited in surface layers of the capsules by suspending them in a .005~
~w/v) aqueous solution of poly L lysine, (MW 43,000 daltons) for 3 minutes.
Th~ resulting ~apsules are suspended in CMLR-1969 P~upplemented with 10% fetal calf serum. The foregoing steps are all conducted at 37C. After incubation at the same tempera~ure, the capsules, if examined under the microscope, will be found to -lg-1 contain fibrobla~ts which have undergone mito~is and display thr~e-dimensional fibroblastic morphology within the microcap-sules.
Selective Dlsru~tion of the Membranes Exa~le 5 ._ Microcapsules from any of examples 1-4 may be treated as follows in order to ~electively disrupt the capsule membranes without damage to the encapsulated core material.
10 ml por~ions of microcapsule suspensions containing about 500-5000 capsules per ml are allowed ~o se~tle and the suspension mediurn is aspirated ofq The capsules are washed twice with saline. The washed capsules are then mixed with a 300 ml aliquot of saline con~aining heparin in various concentrations a~ set forth below and 1.1% (w/v) CaC12. Capsules having algi-nate enclosed therewithin, on completion of this step, display a c3elled, ~hape-retairling interior core. The ~uspension i8 agi-tated at 37C for 10 minutes, after which the capsules are allowed to settle, the supernatent is a~pirated off~ and the cap-sules are washed twice with 3.0 ml of 0.15M NaCl. After aspira-tion of the second wash solution, the capsules are mixed with 2.0 ml of a mixed solution comprising equal volumes of 110 mM sodium citrate and 0.15 M NaCl (pH~-7~4).
Capsule membranes which had been treated with 1,000 u~its/ml heparin and vortexed in the NaCl-NaCitrate solution for 1 minute were completely disintegrated~ The same reault is achieved with capsules treated with 2,000 units/ml hep~rin for 2 rninutes, followed by 15-30 seconds o hand vortexingO Lower ~f~
1 concentrations of heparin are preferred as the possibility of cell damage is decreased.
Af~er dissolution of the mer~ranes any membrane debris may be removed by aspiration and washing. After ~he released cells are resuspend~d in cul~ure medium, they may be tested by the ~ryptan hlue dye exclusion technique and will be found to be in a healthy, viable conditionl with relatively few cells exhi-biting dye up~ake.
Capsules produced in accordance with example 3 are treated, ater washing, with a 3.0 ml solution containing 1,000 units/rnl heparin and 1.0~ AlC13 for 6 minutes with agi~ation.
After aspira~ion of the supernatant, the core material is released by vortexing the capsules wi~h a O.lM solution of sodium ci~rate for 30-90 seconds.
'rhe procedure of example 6 i 8 repeated except that O.lOM EDTA (sodiurn form~ at a pH of 7.0 is used in place of the sodium citrate, resulting in rapid disruption of the capsule membranes.
Example 8 Cap~ules produced in accordance with example 3 are treated, after washing, with a 3.0 ml aqueous solution containing 10 mg/ml of polyvinyl ~ulate (mw approximately 50,000 daltons) and 1~ CaCl~. Post tre~tment with O.lOM sodium citrate results in essentially cornplete dissolution o the capsules.
~21-Other embodimentg are within ~he following claims.
What is claimed is:
BackgroUnd This invention relates to a method of encapsulation which is reversible, that is, a method which may be used to encapsulate a liquid or a solid material and thereafter to release the material by selectively disrupting the capsule membranes~ An important embodimen-t of the invention involves the microencapsulation of living cells which may subsequently be released from within the produced capsule membranes without damage.
Canadian Application Serial No. 348,524, now Patent No. 1,145,258, entitled "Encapsulation of Viable Tissue and Tissue Implantation Mlethod", by F. Lim discloses a micro-encapsulation technique which can be used to encapsulate esscntially any solid or liquid material within semipermeable or substant:ially impermeable capsule membranes. An outstan~ing advantage of the process is that the conditions under which th~ capsule membranes are formed involve no toxic or denatur-.ing reagents, ~xtremes of temperature, or other conditions which damacJe living cells. The process of that application is ~ according:ly well-suited for the production of microencapsulated l:Lving materials which remain viable and in a healthy state.
Because the process allows a degree of control of the perme-~bility o~ the membrane, it is now possible to microencapsulate cell cultures of procaryotic, eukaryotic~ or other origin such that cells of the culture are protected from contaminating bacteria, high molecular weight immunoglobulins, and other potentially deleterious factors, and remain confined within a microenvironment well-suited for their continuing viability and ongoing metabolic functions. If the ~sj D~
1 microcapsules are suspended in a conventional culture medium suf-ficient to suppo~t growth of the living cells involved, the microencapsulated cells are free ~o ingest substances needed for metabolism which diffuse through the membrane and to excrete their me~abolic products through the capsule membrane into the surrounding medium.
5~
1 Summar~ of the Invention The instant invention is directed to a method of selec-tively disrupting certain membranes synthesized during the micro-encapsulation procedure se~ forth in the above-referenced application without any detectable damage to the encapsulated core rnaterial, ~ore specifically, the process of this invention is practiced on membranes comprising a water-insoluble matrix formed froTn at least two water-soluble componen~s: a polyme_ which includes multiple cationic moieties [polycationic polymer, e.g. polye-thylene amine); and a polymer having multiple anionic rnoieties (polyanionic polymer, e.g. sodium alginate gum). The two components are connected by ~alt bridges between the anionic and cationic moieties to form the matrix.
The process of the invention comprises the steps of expo~ing membranes of the type set forth above to a solution of ca~ions, peferably, monatomic or very low molecular weight multi-~alent cations/ and a solution of a stripping polymer having plural anionic rnoieties. Preferably, these solutions are mixed together. The anionic charge of the polymer ~hould be sufficient to disrupt the salt bridges and should preEerably be equal to or yreater than the charge density of the polyanionic polymer of the membrane. The solution is contacted with the capsules to allow the cations, e.g., calcium or aluminum, to eornpete against the polycationic polymer in the capsule m~m~rane for anionic sites on the polyanionic polymer. Sirnul~aneously, the stripping polymer having plural anionic moieties competes with the polyanionic polymer for cationic sites on the polymer chains~ This results ; in "softening" or "unæipping" of the capsule membranes. To 1 complete the disruption, the capsules are wa8hed a~d then exposed to a sequestering agent to r~move cations associated with the polyanionic polymer~ The preferred sequestering agents are che-lating ayents such as citrate ion~ or EDTA ions. If~ as in an important embodiment, the capsules con~ain viable cells, it is preferred to mix the sequestering agent with an isotonic saline solution.
The currently preferred cation is calciumO Examples of the stripping polymer having plural anionic yroups used in the mixed solution include polysulfonic acids or (pre~erably) their salt~, either natural or synthetic. Outstanding results have been obtained using heparin, a natural polymer containing plural ~ulfonate groups. Polymers containing polyphosphoric or polyacrylic acid salt moie~ies may also be used. The currently preferred seques~ering agent is sodium citrate.
The invention also contemplates a method of encap-sulati.ng viabLe cells within a pro~ective environment and sub-sequently releasing the cells. Thus, the invention provides what may be described as a package which maintains living cell cultures of whatever origin in a sterile, stablizing environment in which they can undergo normal metabolism and even mitosis and from which they subsequently can be released.
Accordingly, an object of the invention is to provide a method for encapsulating living cells within permeable membranes and subsequently selectively disrupting the membranes to release the cells. Another object is to provide a process for selec-tively disrupting membranes. Another object is to disrupt mem-1 branes without damage to proximate living tissue. Still anotherobject is to provide a method of encapsulatiny and subsequently releasing finely divided materials J liquids J and solutions.
These and other objects and features of the invention will be apparent from the following description of some important embodirnents.
Description The selective membrane disruption process of the inven-tion is practiced on membranes consisting of a salt bridge-bonded ]0 matrix of a polycationic polymer and a polyanionic polymer.
U~ually, the membranes will have a spheroidal form defining an enclo~ed int~rior containing an encap~ulated substanceO However, ~he proce~s may al50 be practiced on membranes o~ this type which take other than spheroidal form, Although es~entially any material ~compatible with aqueous environments) in liquid or solid form can be encapsulated and subsequently released without damage by the process of this irlvention, its most notable utility, as prPsently contemplated, lies in it~ abili~y ko encapsulate and subsequently release living systems such as cell cultures. Accordingly, the descrip-tion which follows will be primarily confined to a discussion of the encapsulation and release of cell~. Those skilled in the art will be able to adap~ ~he process withou~ difficulty to the encapsulation of less fragile materials.
lation __.
The tissue or cells to be encapsulated are suspended in an aqueous medium ~uitable for maintenance and for supporting the 1 ongoing me~abolic processes of the particular cell type involved.
Media sui~able for this purpose are well known to those sXilled in the art and often ar~ availahle commercially. The average diameter of the cell mass or ot~er material to be encapsula~ed can vary widely between a few microns to a millimeter or more.
Mammalian Islets of Langerhans, for examples, are typically 50 to 200 rnicrons in diameter. Tissue fragments and individual cells such as fibroblasts, leukocytes, lymphoblastoids, pancreatic beta, alpha or delta cells, islet of Langerhans, hepatocytes, or the cells of other tisgue may be encapsulated as desired~ Also, microorganisms may be encapsulated including those which have been genetically modified by recombinant DNA or other techniques, The ongoing viability of such living matter is depen-dent, inter alia, on the availability o~ required nutrients, oxy-gen transfer, absence of toxic substances in the medium, and the pH o~ the medium. Heretofore, it has not been possible to main-tain suc~l living rnatter in a physiologically compatible environ-ment while simultaneouæly encapsulating, The problem has been that the conditions required for membrane formation have been ~0 l~tllal or harmful to the tifisue, and prior to the invention of the above-referenced application, no method of membrane ormation which allowed tissue ~o survive in a healthy state had been forthcoming.
However, it has been discovered that certain water-~oluble substances which are physiologically compatible with living tissue and can be rendered water-insoluble to form a æhape-retainingO coherent mass, can be used to form a "temporary cap~ule" or protective barrier layer about individual cells, --7~
l groups of cells, or tissues. Such a subs~ance is added, typically at a concen~ration on the order of 1-2 weight percent to the tissue culture medium. The solution is then formed into droplets containing tissue together with its maintenance or growth 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 more permanent membrane which, in accordance with this invention, may be subsequently ~electively disrupted to release the encapsulated tissue without damage. Where the ma~erial used to form the temporary capsules permi~s, the capsule interior may be reliquified ater formation of the permanent membrane. This is done by re-establishing the conditions in the medium at which the ma~erial is soluble.
The material used to form the temporary capsules may be any non-toxic, water-soluble material which, by a change in ~he ~urrounding io~ic environment or concentration, can be converted to a shape-retaining mass. The material also comprises plural, easily ionized anionic moieties, e.g., carboxyl grc,ups, which can react by sal~-bond forrnation with polymers containing plural cationic groups. As will be explained below, this type of material enables the d~position of the permanent membrane of a selected permeability (including substantially non-porous to a level of several hundred thousand daltons).
The presently preferred polyanionic material for forming the temporary capsule ars acidic, water-soluble, natural or syIl~hetic polysaccharide gumsO Many such materials are com-mercially available~ They are typically extracted from vegetable 1 matter and are often used as additives ~o various foodsO Sodium alginate i3 the presently preferred anionic polymer. Alginate in the molecular weigh~ range of 150,000~ daltong may be used, but because of its molecular dimensions will usually be unable to permeate the finally formed capsule mernbranes. To make capsules without trapped liquid alginate, lower molecular weight alginate, e.g., 40,000--80,000 daltons can be used. In the inished cap-sule, the alginate can then be more easily removed from the intracapsular volume by diffusion through a membrane of suf-ficient porosity. Other useable polyanionic yums include acidic fractions of guar gum, carageenan, pectin, tragacanth gum, or x~nthan gurQ.
These materials compriee glycoside-linked saccharide chain~. Their free acid groups are often present in the alkali m~tal ion form, e.g., ~odium form. If a multivalent ion such as calcium, strontium, or aluminum is exchanged for the alkali metal ion, the liquid, water-soluble polysaccharide rnolecules are "croas-linked" to forrn a water-in~oluble, shape retaining gel which can be resolublized on rernoval of the ions by ion exchange or via a suquestering agent. While essentially any multivalent ion which can form a salt with the acidic gum is operable, it is preferred that physiologically compatible ions, e.g., calcium, be employed. This tends to preserve the tissue in the living state.
Other multivalent cations can be used for less fragile materialO
Magrlesiurn ions are ineffective in gelling sodium alginate.
A typical solution composition comprises equal volumes o a cell suspensiorl in its medium and a one to two percent solu-tion of gum in physiological saline. When employing sodium algi-nate, a 1.2 to 1.6 percent solution has been used with success.
1 In the next s~ep of the encapsulation process, the gum solution containing the tissue i5 formed into droplets of a desired size sufficient to envelop the cells ~o be encapsulated.
Thereafter, the droplets are immediately gelled to form shape-re~aining spherical or spheroidal masges~ The drop formation may be conducted by known techniques.
A tube containing an aqueous solution of rnultivalent cations, e.g., 1.5~ CaC12 solution, is fitted with a stopper which holds a drop forming apparatus. The appartus comprises a housing having an upper air intake nozzle and an elongate hollow body friction fitted into the stopper. ~ lO cc syringe equipped with a stepping pump is mounted atop the housing with, e~g., a O.Ol inch I.D. Teflon coated needle passing through the length of the housing. The interior of the housing is designed such that the tip of the needle is subjec~ed to a constant laminar air flow which acts as an air knife. In use, with the syringe full of ~olution cont~ining the rnaterial to be encapsulated, the stepping pump i~ actuated to incrementally force droplets of solution from the tip o~ the needle~ Each drop is "cut of-f" by the air stream and fall~ approximately 2.5 cm into the CaC12 solution where it is immedia~ely gelled by absorption of calcium ions. The distance between the tip of the needle and the surface of the CaC12 solution is great enough, in this instance, to allow the sodium algina~e/cell suspension to assume the most physically favorable æhape; a æphere (maximurn volume for minimwn surface area). Air within the tube bleeds through an opening in ~he stopper. This results in "cross-linking" of the gel and in the formation of a high viscosity, shape-retaining protective tem-1 porary capsule containing the suspended ti~sue and its medium.The capsules collect in the solution as a separate phase and are separated by aspiration.
In the next step of the proc~ss, a membrane is deposited about the eurface of the temporary capsules by cxoss-linkiny surface layers. This is done by subjecting the temporary cap~ules comprising polyanion to an aqueous solution of a polymer containing cationic groups reactive with anionic functiorlalities in the polyanionic polymer. Polymers containing reactive cationic groups such as free amine groups or combina~ions of amine and imine groups are preferred. In this situation/ the polysaccharide gurn is crosslinked by interaction (sal~ bond ~ormati.on) between the carboxyl groups and the amine or imine groups of the polycationic polymer. Advantageously, permeability can be controlled within limi~s by selecting the molecular weight o~ the cro~s-linking polymer used and by varying exposure time and the concentration of poLymer in solution. A solution of polymer having a low molecular weight, in a given time period, will penetra~e further into the temporary capsules than will a high molecul~r weight polymer. The degree of penetration of the cross-linker has been correlated with the resulting permeability.
In general, the higher the molecular weight and the less penetra-tion, the larger ~he pore size. Longer exposures and more con-centrated polymer solutions tend to decrease the resulting mernbrane'~ upper limit of permeability However, the average molecular weight of the polymer is the dominarlt determinant.
Broadl~, polymers within the molecular weight range o~ 10,000 to 100,000 daltons or greater may be used, depending on the duratio 1 of the reaction, th~ concentration of the polymer solution, and the degree of permeability desirzd. One successful ~et of reac-tion conditions, using polylysine of average molecular weight of about 35,000 daltons, involved reaction for three minutest with stirring, o a physiological saline solution containing 0~0167 percent polylysine~ This results in membranes having an upper limit of permeability of about lOOrOOO daltons. Generally, higher molecular weight materials form membrane which are more difficult to subsequently disrupt as compared with lower molecular weight ma~erials. The charge densi~y of the crosslinking polycationic polymer also affects the pore size and ea~e of membrane disruption. Generally, higher charge density materials form less porous membranes which are more di~ficult to dlsrupt. Optimal reac~ion conditions suitable for controlling permeability in a given sys~em can readily be determined emp~rically in view of the foregoing guidelines.
Example~ of suitable cross-linking polymers include proteins and polypeptides, either natural or synthetic, having ~ree amino or cornbinationa of amino and imino groups, polyethyl-enearnines, polyethyleneimines, and polyvinylamines Polylysine,in both the D and L forms, has been used with success. Proteins such as polyarginine, polycitrulline, or polyornithine are also operableO Polymers in the higher range of positive charge den-sity (e.g., polyvinylamine) vigorously adhere to the anionic groups of the polyanionic molecules and are rnore difficult to disrupt.
At this point in the encapsu]ation, capsules may be collected w~ich cornprise a "psrmanent" semipermeable membrane 1 surrounding a gelled solution of gum, cell type compatible culture medium, and the cells. If the obje~t is simply to preserve the cells in a protective environment, no further steps need be done. However, if mass transfer is to be promoted within the capsules and across the membranes, it i6 preferred to re~lj.quify the gel to its water- oluble form, This may be done by reestahlishing the conditions under which the gum is a liquid, e.g., removing the calcium or other multifunctional ca~ions from the gel. The medium in the capsule can be resolubilized simply by immersin~ the capsules in phosphate buffered saline, which contains alkali metal ions and hydrogen ions. Monovalent ions exchange with the calcium or other multifunc~ional ions within the gum when the capsules are immersed in the solution with stirring. Sodiurn citra~e solu~ions may be used for the same purpose, and serve to sequester the divalent ions~ Gum molecules having a molecular weight below the upper limit of permeability o~ th~ rnernbranes may subsequently be removed from the intracap-sular volurne by diffusion.
La3tly, it may be desirable to -treat the capsules so as to tie up free amino groups of the like which might otherwise impart to the cap~ules a tendency to clump~ This can be done, for example, by immersing ~he capsules in a dilute solution of sodium alginate.
From the foregoing it will be apparent that no harsh reagents, extremes of tempera~ure, or other conditions deleterious to the health and viability of the cells need be used in the membrane formation process. Thuss even very sensitive cells such as mammalian hepatocytes, leukocytes, ~ibroblasts, 1 lymphoblasts, and cells from various endocrine ~issues may be encapsulated withou~ difficul~y~ Of course, ce~ls of microbial oriyin such as yeasts, molds, and bacteria which are bet~er adapted to survive in hostle environtnentsl as well as inert reagents, solids, or biologically active materials may also be encapsulated without damage.
Encapsulated cells of the type described above may be suspended in maintenance medium or growth medium for storage or culture and will remain free of bacterial infection. If suspended in growth medium, cells which undergo rni~osis ln vitro will do so within the capsules Normal in vitro metabolism con-tinues provided the faetors needed for metabolic processes are of ~u~iciently low molecular weight that ~hey can penetrate the capsule mellbrane, or are encapsulated together with the cells.
Metaboli.c products of the cells (if of a molecular weight below the upper limit of permeability) penetrate the membrane and ~ollect in the medium. The cells in encapsulated form may be stored, shipped, or cultured as desired, and may be released from their protective environmen~ without damage by means of the following process of selectively disrupting the membranes.
Disruption oi Membrane In accordance with the invention the encapsulated material may be released by a two step process involving commer-cially available reagents having properties which do not adversely affect the encapsulated cells.
Eirst, the capsules are separated from their suspending medium, washed thoroughly to remove any contaminants and then s~
1 dispersed, with agitation, in a separate or preferably mixed solution o~ cations such as calcium ions or ot~er monatomic (low molecular weigh~ multivalen~ cation) and a stripping polymer having plural anionic moieties such as polysulfonic acid groups~
Polymers containing polyphosphoric or polyacrylic acid moieties ma~ also be used. Heparin, a natural sulfonated polysaccharide, is preferred ~or disrupting membranes containing cells. The anionic charge of the stripping polymer used must be sufficient to diRrupt the salt bridges. Thus the anionic charge density may be equal to or preferably greater than the charge density of th~
interior polyanionic polymer (e.g~, ~he gum) originally employed to forrn the membranes. The molecular weight of the stripping polymer should be at least comparable to and preferably greater than the molecular weight of the interior polycationic polymer used in forrning the merabrane. Within the suspension, the calcium ions com~ete with the interior polycationic polymer comprising the membrane for anionic sites on the polyanionic polymer.
5imultaneously, the stripping polymer dissolved in the solution competes wi~h the polyanionic gum in the membrane for cationic ~0 site~ on the polycationic polymer. This results in a water-dispersible or preferPably water-soluble complex of, e.g~, poly-lysine and the polyanionic polymer, and in association of the cations with gel molecules.
This step renders the membrane susceptible to subsequent exposure to a sequestering agent which completes the disruption process by taking up di or trivalent ions from the gel. Typically, capsule membrane debris, if any, which remains in the medium can be separated easily from the cells.
1 The currently preferred ~olution for the first stage of the selective disruption process comprises 1.1~ calcium cloride (w/v) and between 500 to 1,500 units of heparin per milliliter of solu~ion. A volume of microcapsules is added ~o this solution sufficient to constitute between about 20~ and 30~ of the total volume of suspensionO Calcium chloride and heparin are preferred when disrup~ing membranes of cell-containing capsules since both reagents are physiologically compatible with most cells and mini-mize the possibility of cell damage. Mixtures of aluminum salts or other multivalent cations ~but not Mg+~ ions) may also be used together with the polysulfonic or other acid salt of the type set ~orth above.
In general, the concentration of the ions ~nd anionic polymer in the solution used in this step may vary widely.
Optimum concentrations may be readily determined empirically.
The lowe~t operable concentration for a particular batch o~
~ncap~ula~ed cell~ is preferred.
The curren~ly preferred sequestering agent for per-forming the selective disruption is sodium citrate, although other allcali metal citrate salts and alkali metal EDTA may also be used. When sodium citrate is employed, the optimum con~
centration is on the order of 55 mM. Where the cap~ule membranes being disrupted contain viable tissuel it is preferred that the citrate be dissolved in isotonic saline so as to minimize cell darnageO
The inverltion will be further understood from the following non-limiting examples.
1 Capsule Formation Exam~le_l: Encapsulat.ion of Panc eatic Tissue Islets of Langerhans are obtained rom rat pancreas and added to a complete tissue culture (C~RL-1969 Connaught Laboratories, Toronto, Canada) a~ a concentration of approxima-tely 103 i51et3 per rnilliliter, The tissue culture contains all nutrients needed for continued viability of the islets as well as the amino acids employed by the cells for making hormones. Four-tenths of a milliliter of an islet suspension containing approxi-mately 103 islets per milliliter is then added to a one-half milliliter volume of 102 percen~ sodium alginate ~Sigma Chemical Company) in physiological saline.
Next, a 1.5 percent calcium chloride solution is used to gel droplets on the order of 300-400 microns in diameter.
After the supernatant solution is removed by aspiration, the ~lled droplets are transferred to a beaker containing 15 ml o a ~olution cornpri~ing one part of a 2% 2 (cyclohexylamino) ethane sulfonic acid buffer solution in 0~6~ NaCl (isotonic, pH-8,2) diluted with 20 parts 1% CaCl~. After a 3 minute immersion, the capsules are washed twice in 1% CaC12.
The capsules are then transferred to a 32 ml solution comprising 1/80 of one percent polylysine (average MW 35,000 amu~
in physiological saline. After 3 minutes~ the polylysine solu~
tion is decantedO The capsules are washed with 1% CaC12, and optionally re~uspended for 3 minutes in a solution of polyethyl-eneimine (MW 40,000-60,000) produced by diluting a stock 303~
polyethyleneimine solution in morpholino propane sulfonic acid bufer (0.2M, pH=6) with sufficient 1% CaCl~ to result in a final 1 polymer concentration of 0 D 12~. The re~u~ting capsules, having "permanent" semipermeablP membranes, are then washed twice with 1% CaCl ~, twice with physiological saline/ and mixed with 10 ml of 0.12 percent alginic acid solution.
The capsules resist clumping, and many can be seen to contain islet~ of Langerhans. Gel on the in~erior of the cap-sules is reliquified by immersing th~ capsules in a mixture of ~aline and citrate buffer (pH-7~4) for 5 minutes~ Lastly, the capsules are suspended in CMLR-69 medium.
Under the microscope, these capsules are observed to compri e a thin membrane w~ich encircles an islet within which individual cells can be seen. Molecules having a molecular weight up to about one-hundred thousand can traverse the membra-nes. This allows oxygen, amino acids, nutrients, and plasma com-pon~nts used in cul~ure media (e.g,, lower molecular weight fetal cal~ serurn co~nponents) ko reach khe islek and allows insulin to be excreted.
Exam~le 2. _Encapsulation of Hepatocyte~
The procedure of example 1 is repea~ed except ~ha~ 0.5 ml of a liver cell suspension containing about 105 cells per milliliter is used in place of the 0.4 ml islet suspension. The ongoing viability of the liver cells has been demonstrated by the dye exclusion technique (~rypan blue exclusion) and by their observed ability to con~inuously produce urea. It is known that liver tissue, in vitro, can ingest toxins from its environmen~.
Accordingly, toxins of a molecular weight low enough to pass t~rough khe semipermeable membranes are detoxiied by the cells.
1 Example 3_ Activated Charcoal Encaps la~ion The procedure of exarnple l is repeated except that par-ticulate activated charcoal is suspende~ directly in the sodium alginate solution, the half milliliter of tissue suspension is omitted, and polylysine of of an average rnolecular weight of 35,000 is used as a cross-linkerG As long as the charcoal par-ticles are smaller than the smallest inside diameter of the capi.llary used to produce the dropl~ts, charcoal of high surface area surrounded by a semipermeable membrane resultsO These effectively prohibit the escape of charcoal chips or dust, yet can be used to absorb materials of any pre-selec~ed molecular weight ranye from fluid passed through the capsules~
4: Enca~sulation of Human Fibroblasts Human fibroblasts obtained by treating human foreskin tiRsue with trypsin and EDTA for 5 minutes at 37C in a known manner are suspended in a complete growth medium (CML,R 1969, C~nnaught Laboratories) ~upplemented with 40% (v/v) puriEied fetal calf ~erurn, 0.8% sodium alginate (Sigma) and 200 mg/ml puri1ed cal~ skin collagen. The density of the cell suspension is about 1.5 x 107 cells/ml. Temporary alginate capsules are formed as set forth abovel Semipermeable membranes are deposited in surface layers of the capsules by suspending them in a .005~
~w/v) aqueous solution of poly L lysine, (MW 43,000 daltons) for 3 minutes.
Th~ resulting ~apsules are suspended in CMLR-1969 P~upplemented with 10% fetal calf serum. The foregoing steps are all conducted at 37C. After incubation at the same tempera~ure, the capsules, if examined under the microscope, will be found to -lg-1 contain fibrobla~ts which have undergone mito~is and display thr~e-dimensional fibroblastic morphology within the microcap-sules.
Selective Dlsru~tion of the Membranes Exa~le 5 ._ Microcapsules from any of examples 1-4 may be treated as follows in order to ~electively disrupt the capsule membranes without damage to the encapsulated core material.
10 ml por~ions of microcapsule suspensions containing about 500-5000 capsules per ml are allowed ~o se~tle and the suspension mediurn is aspirated ofq The capsules are washed twice with saline. The washed capsules are then mixed with a 300 ml aliquot of saline con~aining heparin in various concentrations a~ set forth below and 1.1% (w/v) CaC12. Capsules having algi-nate enclosed therewithin, on completion of this step, display a c3elled, ~hape-retairling interior core. The ~uspension i8 agi-tated at 37C for 10 minutes, after which the capsules are allowed to settle, the supernatent is a~pirated off~ and the cap-sules are washed twice with 3.0 ml of 0.15M NaCl. After aspira-tion of the second wash solution, the capsules are mixed with 2.0 ml of a mixed solution comprising equal volumes of 110 mM sodium citrate and 0.15 M NaCl (pH~-7~4).
Capsule membranes which had been treated with 1,000 u~its/ml heparin and vortexed in the NaCl-NaCitrate solution for 1 minute were completely disintegrated~ The same reault is achieved with capsules treated with 2,000 units/ml hep~rin for 2 rninutes, followed by 15-30 seconds o hand vortexingO Lower ~f~
1 concentrations of heparin are preferred as the possibility of cell damage is decreased.
Af~er dissolution of the mer~ranes any membrane debris may be removed by aspiration and washing. After ~he released cells are resuspend~d in cul~ure medium, they may be tested by the ~ryptan hlue dye exclusion technique and will be found to be in a healthy, viable conditionl with relatively few cells exhi-biting dye up~ake.
Capsules produced in accordance with example 3 are treated, ater washing, with a 3.0 ml solution containing 1,000 units/rnl heparin and 1.0~ AlC13 for 6 minutes with agi~ation.
After aspira~ion of the supernatant, the core material is released by vortexing the capsules wi~h a O.lM solution of sodium ci~rate for 30-90 seconds.
'rhe procedure of example 6 i 8 repeated except that O.lOM EDTA (sodiurn form~ at a pH of 7.0 is used in place of the sodium citrate, resulting in rapid disruption of the capsule membranes.
Example 8 Cap~ules produced in accordance with example 3 are treated, after washing, with a 3.0 ml aqueous solution containing 10 mg/ml of polyvinyl ~ulate (mw approximately 50,000 daltons) and 1~ CaCl~. Post tre~tment with O.lOM sodium citrate results in essentially cornplete dissolution o the capsules.
~21-Other embodimentg are within ~he following claims.
What is claimed is:
Claims (14)
1. A process for selectively disrupting a permeable capsule membrane comprising a matrix of a first polymer having multiple cationic moieties; and a second polymer having multiple anionic moieties and a first charge density, said first and second polymers being connected by salt bridges between said anionic and cationic moities;
said process comprising the steps of: .
A. exposing said membrane to a solution of cations and a stripping polymer having plural anionic moieties, said stripping polymer having sufficient charge to disrupt said salt bridges;
B. allowing said cations to compete with said first polymer for anionic sites on said second polymer, and said stripping polymer to compete with said second polymer for cationic sites on said first polymer; and C. sequestering cations associated with said second polymer after step B.
said process comprising the steps of: .
A. exposing said membrane to a solution of cations and a stripping polymer having plural anionic moieties, said stripping polymer having sufficient charge to disrupt said salt bridges;
B. allowing said cations to compete with said first polymer for anionic sites on said second polymer, and said stripping polymer to compete with said second polymer for cationic sites on said first polymer; and C. sequestering cations associated with said second polymer after step B.
2 . The process of claim 1 wherein said sequestering step (C) is effected by exposing said membranes to a solution containing a chelating agent.
3. The process of claim 2 wherein said chelating agent is selected from the groups consisting of citrate ions and EDTA
ions.
ions.
4. The process of claim 1 wherein said first polymer comprises multiple amino groups.
5. The process of claim 4 wherein said second polymer comprises an acidic, water-soluble gum.
6. The process of claim 5 wherein said second polymer comprises alginate.
7. The process of claim 4 wherein said stripping polymer comprises heparin and said cations comprise Ca++.
8. The process of claim 1 wherein said first polymer is selected from the group consisting of:
a) proteins comprising plural amino acid units having free amino groups;
b) proteins comprising plural amino acid units having free imino groups;
c) polypeptides comprising plural amino acid units having free amino groups;
d) polypeptides comprising plural amino acid units having free imino groups;
e) polyvinyl amines;
f) polyethyleneimines;
g) polyethyleneamines; and h) mixtures thereof.
a) proteins comprising plural amino acid units having free amino groups;
b) proteins comprising plural amino acid units having free imino groups;
c) polypeptides comprising plural amino acid units having free amino groups;
d) polypeptides comprising plural amino acid units having free imino groups;
e) polyvinyl amines;
f) polyethyleneimines;
g) polyethyleneamines; and h) mixtures thereof.
9. The process of claim 1 wherein said stripping polymer is selected from the group consisting of:
a) polysulfonic acids;
b) polyphosphoric acids;
c) salts thereof; and d) mixtures thereof.
a) polysulfonic acids;
b) polyphosphoric acids;
c) salts thereof; and d) mixtures thereof.
10. The process of claim 1 wherein said stripping polymer is a polysulfonic acid salt polymer.
11. The process of claim 1 wherein said stripping polymer having plural anionic moieties has a charge density greater than said first charge density.
12. The process of claim 1 wherein said membrane defines a microcapsule containing a living cell.
13. The process of claim 12 wherein said first polymer comprises a protein, said second polymer comprises sodium alginate, said cations comprise calcium, and said stripping polymer having plural anionic moieties comprises heparin.
14. The process of claim 13 wherein said sequestering step is effected with citrate dissolved in a solution physiologi-cally compatible with said cell.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US24358481A | 1981-03-13 | 1981-03-13 | |
| US243,584 | 1981-03-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1184518A true CA1184518A (en) | 1985-03-26 |
Family
ID=22919328
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000398210A Expired CA1184518A (en) | 1981-03-13 | 1982-03-12 | Reversible microencapsulation |
Country Status (11)
| Country | Link |
|---|---|
| JP (1) | JPS57197031A (en) |
| BE (1) | BE892477A (en) |
| CA (1) | CA1184518A (en) |
| CH (1) | CH651579A5 (en) |
| DE (1) | DE3209045C2 (en) |
| DK (1) | DK112382A (en) |
| FR (1) | FR2501528A1 (en) |
| GB (1) | GB2094750B (en) |
| IT (1) | IT1150681B (en) |
| NO (1) | NO158284C (en) |
| SE (1) | SE454181B (en) |
Families Citing this family (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DD160393A3 (en) * | 1980-11-14 | 1983-07-27 | Horst Dautzenberg | MICRO CAPSULES AND METHOD FOR THE PRODUCTION THEREOF |
| NO161446C (en) * | 1981-03-13 | 1989-08-16 | Damon Biotech Inc | PROCEDURE FOR CULTING CELLS RELATED TO ANCHORING. |
| NO163060C (en) * | 1981-03-13 | 1990-03-28 | Damon Biotech Inc | PROCEDURE FOR THE PREPARATION OF SUBSTANCES MADE BY LIVING CELLS PRODUCING INTERFERONES OR ANTIBODIES. |
| US4582799A (en) * | 1983-04-15 | 1986-04-15 | Damon Biotech, Inc. | Process for recovering nonsecreted substances produced by cells |
| CA1196862A (en) * | 1983-06-01 | 1985-11-19 | Anthony M.F. Sun | Microencapsulation of living tissue and cells |
| EP0127989A3 (en) * | 1983-06-01 | 1986-03-26 | Connaught Laboratories Limited | Microencapsulation of living tissue and cells |
| IL72787A (en) * | 1983-09-01 | 1987-12-31 | Damon Biotech Inc | Polyionic microencapsulation of viable cells |
| US4663286A (en) * | 1984-02-13 | 1987-05-05 | Damon Biotech, Inc. | Encapsulation of materials |
| DE3575375D1 (en) * | 1984-02-15 | 1990-02-22 | Massachusetts Inst Technology | ENCLOSURE METHOD AND SYSTEMS WITH ENCLOSED ACTIVE MATERIAL. |
| US4686098A (en) * | 1984-05-14 | 1987-08-11 | Merck & Co., Inc. | Encapsulated mouse cells transformed with avian retrovirus-bovine growth hormone DNA, and a method of administering BGH in vivo |
| GB8500121D0 (en) * | 1985-01-03 | 1985-02-13 | Connaught Lab | Microencapsulation of living cells |
| EP0213908A3 (en) * | 1985-08-26 | 1989-03-22 | Hana Biologics, Inc. | Transplantable artificial tissue and process |
| JPS62166891A (en) * | 1986-01-20 | 1987-07-23 | Snow Brand Milk Prod Co Ltd | Production of useful substance by cell cultivation |
| US4952506A (en) * | 1986-04-28 | 1990-08-28 | Rohm And Haas Company | Immobilization of nonanchorage-dependent cells |
| JPS6348039U (en) * | 1986-09-12 | 1988-04-01 | ||
| JPS6379587A (en) * | 1986-09-22 | 1988-04-09 | Saburo Senoo | Base material for cultivating cell |
| US5069831A (en) * | 1988-12-22 | 1991-12-03 | The Mead Corporation | Method for separation of microcapsules and preparation of printing inks |
| WO1992019195A1 (en) * | 1991-04-25 | 1992-11-12 | Brown University Research Foundation | Implantable biocompatible immunoisolatory vehicle for delivery of selected therapeutic products |
| US5800829A (en) * | 1991-04-25 | 1998-09-01 | Brown University Research Foundation | Methods for coextruding immunoisolatory implantable vehicles with a biocompatible jacket and a biocompatible matrix core |
| IL102785A (en) * | 1991-08-20 | 1998-06-15 | Univ Leicester | Method for removing protein from a water- soluble gum and a method of making biocompatible capsules using said gum |
| JP2003516364A (en) * | 1999-12-08 | 2003-05-13 | ウラディミロビチ ズィービン，ドミトリー | Methods of using polyacrylamide gels to form capsules in mammalian biological tissues, cell culture methods, and methods of treating cancer and diabetes mellitus |
| GB201408233D0 (en) | 2014-05-09 | 2014-06-25 | Austrianova Singapore Pte Ltd | Use of polyanionic composition |
| JP7159334B2 (en) * | 2018-09-28 | 2022-10-24 | 富士フイルム株式会社 | CELL STRUCTURE, CELL STRUCTURE MANUFACTURING METHOD, CELL CULTURE METHOD, AND MICROCHANNEL |
| CN114504561B (en) * | 2022-03-01 | 2023-08-11 | 陕西科技大学 | Aqueous coating method for preparing drug osmotic pump preparation |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL129921C (en) * | 1958-12-31 | |||
| FR1542881A (en) * | 1966-10-10 | 1968-10-18 | Ncr Co | Process for preparing tiny polymer-based capsules |
| CH573212A5 (en) * | 1973-06-29 | 1976-03-15 | Feller Marc Rech Tech Et Et Sc | Delayed release agricultural formulations - of herbicide or insecticide absorbed on porous carrier and coed with isolating layer |
| US4352883A (en) * | 1979-03-28 | 1982-10-05 | Damon Corporation | Encapsulation of biological material |
-
1982
- 1982-03-11 NO NO820795A patent/NO158284C/en unknown
- 1982-03-12 JP JP57038239A patent/JPS57197031A/en active Granted
- 1982-03-12 CH CH1574/82A patent/CH651579A5/en not_active IP Right Cessation
- 1982-03-12 BE BE0/207557A patent/BE892477A/en not_active IP Right Cessation
- 1982-03-12 DK DK112382A patent/DK112382A/en not_active Application Discontinuation
- 1982-03-12 FR FR8204243A patent/FR2501528A1/en active Granted
- 1982-03-12 IT IT20139/82A patent/IT1150681B/en active
- 1982-03-12 DE DE3209045A patent/DE3209045C2/en not_active Expired
- 1982-03-12 GB GB8207308A patent/GB2094750B/en not_active Expired
- 1982-03-12 SE SE8201557A patent/SE454181B/en not_active IP Right Cessation
- 1982-03-12 CA CA000398210A patent/CA1184518A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| FR2501528B1 (en) | 1984-06-15 |
| DE3209045C2 (en) | 1986-06-19 |
| DE3209045A1 (en) | 1982-09-30 |
| JPS6152737B2 (en) | 1986-11-14 |
| NO158284B (en) | 1988-05-09 |
| JPS57197031A (en) | 1982-12-03 |
| NO158284C (en) | 1988-08-17 |
| CH651579A5 (en) | 1985-09-30 |
| GB2094750A (en) | 1982-09-22 |
| DK112382A (en) | 1982-09-14 |
| IT1150681B (en) | 1986-12-17 |
| FR2501528A1 (en) | 1982-09-17 |
| NO820795L (en) | 1982-09-14 |
| BE892477A (en) | 1982-07-01 |
| GB2094750B (en) | 1984-08-22 |
| IT8220139A0 (en) | 1982-03-12 |
| SE454181B (en) | 1988-04-11 |
| SE8201557L (en) | 1982-09-14 |
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