the present invention provides a process for encapsulating core materials such as viable eukaryotic or prokaryotic , naturally occurring or genetically modified , cells or tissue culture within a semipermeable membrane . the invention also provides a new type of multi - layer capsule . a gel or temporary matrix is formed about the core material or viable cell by reacting a polysaccharide or glucopolysaccharide containing cationic groups with a multivalent anion . surface layers of the resulting temporary matrix are ionically cross - linked by a polymer containing anionic , preferably carboxyl , groups forming a permanent membrane encapsulating the core material . the interior of the capsule may be reliquified by subjecting the capsule to a solution of low molecular weight polycations . core material , for example , enzymes , hormones , antibodies or viable cells , is suspended in an aqueous medium containing a gellable , cationic group - containing polymer . the preferred gellable polymer is an aminated glucopolysaccharide , most preferably chitosan ( poly - d - glucosamine ). chitosan is formed by acid hydrolysis of chitin ( poly - d - n - acetylglucosamine ), the primary building material of invertebrate exoskeletons . chitosan is a long chain , aminated polymer which is physiologically compatible with most cells . it is only slightly soluble in water but can be readily dissolved in dilute acetic acid . stock solutions of chitosan are prepared by dissolving the chitosan in 0 . 5 molar acetic acid . the acetic acid is removed from the solution by repeated dialysis against phosphate buffered saline ( pbs ). chitosan does not precipitate out of this solution if the solution is kept below 37 ° c . the preferred stock solution , 1 . 4 % chitosan in pbs , has been stored successfully at 4 ° c . for extended periods . chitosan prepared as described above is mixed with the material to be encapsulated , forming a solution or slurry , before it is formed into droplets or other shapes . although solutions having a final chitosan concentration of 0 . 4 % ( w / v ) will gel , optimum gelling is obtained with 0 . 8 - 1 . 2 % ( w / v ) chitosan in isotonic , 125 mm na 2 hpo 4 . droplets of the chitosan / core material suspension can be formed by any conventional droplet - forming apparatus . one such droplet - forming apparatus is described below . a tube containing the suspension is fitted with a stopper which holds a droplet - forming apparatus . the apparatus consists of a housing having an upper air intake nozzle and an elongate hollow body friction fitted into the stopper . a 10 cc syringe equipped with a stepping pump is mounted atop the housing with a needle , e . g ., a 0 . 01 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 subjected to a constant laminar air flow which acts as an air knife . in use , the stepping pump is actuated to incrementally force droplets of solution from the tip of the needle . each drop is &# 34 ; cut off &# 34 ; by the air stream and falls approximately 2 . 5 cm into the gelling solution , preferably a disodium hydrogen phosphate solution , where it is immediately gelled by reaction with the negative ions . the distance between the tip of the needle and the surface of the gelling solution is preferable great enough to allow the chitosan / core material suspension to assume the most physically favorable shape ; a sphere ( maximum volume for a minimum surface area ). air from the tube bleeds through an opening in the stopper . this procedure results in a &# 34 ; cross - linking &# 34 ; of the gel and the formation of a high viscosity shape - retaining protective temporary matrix containing the suspended core material and its medium . the temporary matrices collect in the solution as a separate phase and may be separated by aspiration . the preferred multivalent gelling solution is a 125 mm nahpo 4 solution ; however , monobasic sodium phosphate and sodium sulfate solutions have also produced acceptable temporary matrices . sodium citrate can gel the suspension but best results are obtained with monobasic or dibasic phosphate and sulfate solutions . if the anion level is too low ( e . g ., below approximately 100 mm ), gelling may not occur . temporary matrices formed by this process are collected and washed to remove excess gelling solution . the matrices are then subjected to a coating or cross - linking solution of a polyanionic , preferably polycarboxylated polymer . a preferred cross - linking solution is a 1 % solution of poly - l - aspartic acid or poly - l - glutamic acid , diluted 1 : 15 with 150 mm sodium chloride yielding a final polymer concentration of 6 . 6 × 10 - 4 g / 100 ml . the molecular weight of the poly - l - aspartic or poly - l - glutamic acid can range from 3 , 000 - 100 , 000 daltons or higher , but the 25 , 000 - 60 , 000 dalton range is preferred . a reaction time of 3 - 6 minutes at ambient room temperature has been found to be satisfactory . the following non - limiting examples set forth exemplary procedures for practice of the invention . a stock solution of 1 . 4 % chitosan ( csn - sigma chemical co .) in 14 - 17 mm ( w / v ) isotonic pbs , prepared as previously described , was used in this experiment . different concentrations of csn were tested to determine the optimum csn concentration for temporary matrix formation . in each case , 1 ml samples were formed from the csn stock solution and fetal calf serum ( fcs - flow laboratories ). droplets were produced and introduced into solutions as described below using a droplet - forming apparatus as previously described . table 1 illustrates three different csn concentrations ( w / v ) tested in this experiment , 0 . 6 %, 0 . 8 %, and 1 %. table 1______________________________________ concentrationsample # csn ml fcs ml csn ( w / v ) ______________________________________1 0 . 57 0 . 43 0 . 8 % 2 0 . 42 0 . 58 0 . 6 % 3 0 . 72 0 . 28 1 . 0 % ______________________________________ two gelling buffers were prepared , 110 mm sodium citrate ( sigma ), and 125 mm disodium hydrogenphosphate ( sigma ). in all cases , the temporary matrices formed using these buffered solutions were acceptable , but the sodium citrate buffer produced inadequate gelling of sample no . 1 . while all of the samples formed acceptable temporary matrices in the disodium hydrogen phosphate buffer , sample 3 provided the best gelling . a 1 % solution of poly - l - aspartic acid ( 40 , 000 dalton molecular weight - sigma ), was diluted 1 : 15 with 150 mm sodium chloride ( sigma ) to form a 6 . 6 × 10 - 4 g / 100 ml solution . temporary matrices formed from sample 3 were removed from the gelling buffer , washed repeatedly with isotonic pbs , and resuspended in the poly - l - aspartic acid solution . after six minutes at room temperature in the poly - l - aspartic acid solution , the cross - linking reaction was complete , forming the permanent membranes . the capsules were substantially spherical , about 380 - 480 microns in diameter . some capsules had small tails . capsules formed by this process were not sticky and had no tendency towards clumping . porosity of the capsules was determined empirically to be about 80 , 000 daltons . this example illustrates that acceptable . capsules can be formed using the process of this invention . this example was conducted to demonstrate that the viability of cells is not impaired by the encapsulation process . the cell culture used in this experiment was the friend erythroleukemic cell line ( fel 745 ), a mouse erythroleukemic line which grows readily in a suspension culture . a culture of fel 745 cells was centrifuged for 5 minutes and the resulting pellet was resuspended as a slurry in fetal calf serum ( flow labs ). a stock solution of 1 . 4 % chitosan was prepared as previously described and mixed with the cell culture solution , yielding a final chitosan concentration of 1 % ( w / v ) and a cell concentration of about 5 × 10 6 cells / ml . temporary matrices were produced by forcing the chitosan - cell mixture through a droplet - forming apparatus ( previously described ) and contacting the resulting liquid microspheres with the 125 mm ( w / v ) phosphate ion solution previously described . the resulting temporary matrices were washed repeatedly with isotonic pbs and resuspended in a 6 . 6 × 10 - 4 g / 100 ml ( w / v ) solution of poly - l - aspartic acid ( 40 , 000 daltons molecular weight ) in 150 mm sodium chloride . after six minutes at room temperature in the poly - l - aspartic acid solution , the resulting capsules were washed twice with the culture medium rpmi - 1640 ( flow labs ) containing 10 % fetal calf serum and antibiotics . the microcapsules were placed into tissue culture flasks with the culture medium and incubated at 37 ° c . o at a 5 % co 2 atmosphere . samples of the cell culture were removed at various intervals . examination under a microscope showed that the cells were growing and reproducing , illustrating cell viability after the encapsulation procedure . as may be noted , there was no reliquification step used since friend cells can grow in a gel culture as well as a suspension culture . however , many types of cells need a fluid culture to reproduce . cell viability should not be affected by the resolublization of the capsule interiors . the capsule provides a microenvironment free from external contamination . those skilled in the art may find other variations or embodiments of the process and product described herein within the scope of the invention . acordingly , other embodiments are within the following claims .
disclosed is a process for damage - free encapsulation of a variety of core materials including viable cells . the core material is suspended in an aqueous solution of a polymeric material containing cationic groups such as an aminated glucopolysaccharide , e . g . chitosan . a temporary matrix is formed by gelling droplets of the suspension with a divalent or multivalent anion . the temporary matrix is permanently cross - linked with a polymeric material containing plural anionic groups , e . g ., polyaspartic or polyglutamic acid , to form a semipermeable membrane . the interior of the microcapsule may be resolublized by subjecting the capsule to a solution of low molecular weight cations . the process produces microcapsules which are not sticky , do not clump and allow viable cell growth and proliferation .