CA1172961A

CA1172961A - Process for producing substances produced by cells

Info

Publication number: CA1172961A
Application number: CA000398218A
Authority: CA
Inventors: Franklin Lim
Original assignee: Damon Corp
Current assignee: Damon Corp
Priority date: 1981-03-13
Filing date: 1982-03-12
Publication date: 1984-08-21
Also published as: SE8201554L; DK112282A; GB2094833A; GB2094833B; DE3209127A1; SE454780B; BE892479A; FR2503183A1; NO163060C; NO163060B; JPS6188893A; DE3209127C2; JPS6157288B2; IT1150682B; CH654328A5; FR2503183B1; NO820794L; JPS6244919B2; IT8220140A0; JPS5816693A

Abstract

Abstract of the Disclosure Disclosed is a system and process for producing substances produced in cells such as antibodies and biological response modifiers. Cells which produce the substance of interest are encapsulated within semipermeable membranes having an upper limit of permeability sufficient to allow traverse of ions, amino acids and other cell nutrients and then suspended in a culture medium. Serum components or other high molecular weight materials needed for ongoing viability and normal in vitro metabolism of certain types of cells may be included within the intracapsular volume and may be excluded from the extracapsular medium by limiting the permeability of the membranes. The substance of interest collects either in the intracapsular volume or the extracapsular medium, depending on the degree of permeability of the membranes and on the molecular weight of the substance.

Description

1~7Z~61 1 8ackground of the Invention This invention relates to a method of producing bio-logical material6 of the type generated by cells.

Advance6 in cellular biology have shown that the cells of various higher organisms produce 6mall quantitie6 of substances having significant potential or demon6trable utility for the treatment or diagno6is of disease. Examples of such ; 6ubstances abound in the literature and include various biological response modifiers such as hormones, interferons, and lymphokine6, as well as other sub6tance6 such as antibodies used in diagnostic testing. Cell cultures of microbial origin have long been used to produce large quantities of antibiotics.

Especially in cell cultures derived from higher animal6, there i6 an ever pre6ent danger of bacterial or other contamination. Al60, in most in6tance6 the quantitie6 of the substance of interest produced by cell cultures are very small and collect in the culture medium which contains a complex mixture of serum proteins and other substances. This make isolation and purification of the substance of interest difficult.

~' 1~7~3ti:1 1 SummarY of the Invention This invention provide6 a system and process for pro-ducing substances which are produced by living cells. The practice of the invention has ~he inherent dual advantages of providing a protective environment for the cells of the culture and providing a means of collecting sub6tance6 of interest in a medium having fewer admixed extraneous component~. The invention may be used to separate the 6ubstance of interest from higher molecular weight serum proteins and the like normally required to support the ongoing viability and metabolism of the producing cell6. Alternatively, the invention may be used to collect the substance of interest in a medium containing relatively small quantitie~ of low molecular weight nutrients or cell metabolic products.

The process comprises the steps of encapsulating cells within a membrane which is permeable to the nutrients, ions, and other relatively low molecular weight material~ needed for normal metabolism and ongoing viability of the cell~. The membrane may or may not be permeable to the substance of interest secreted by the cells, but in any case will have an upper limit of permeability sufficient to allow traver6e of molecules having a molecular weight of some selected level generally below about

2.0 x 105 dalton6. The cap6ules 80 produced are suspended in a conventional aqueous culture medium, and the encapsulated cells are allowed to undergo normal in vitro metabolism. Sub6tances of a molecular weight below the upper permeability limit of the membrane which are secreted by the cells permeate the membrane and collect in the culture medium. Advantageou61y, high molecu-lar weight substances ~uch a6 serum protein~ which are required ~L'7;~

1 for health and viability of many types of cell cultures from higher animals, but which typically are themselves not consumed, may be included in the microcapsules where they are confined and prevented from diffusing into the culture medium. Substances which the cell culture consumes during metabolism having a mole-cular weight low enough to permit diffusion through the capsule membranes pa~s therethrough from the culture medium. Metabolic products of the cells having molecula, dimension 6ufficiently small to allow pagsage through the membrane diffuse into the medium external to the capsules. The substances of interest, if of a molecular weight below the upper limit of permeability, dif-fuse into the extracapsular medium where they can be harvested relatively easily because of the absence of contaminating higher molecular weight materials present in prior art unencapsulated cell cultures. If the substance of interest has a molecular weight in excess of the upper limit of permeability of the membranes, then it collects in the capsules which may sub-sequently be isolated from the medium and disrupted for recovery procedures .

The invention is essentially unlimited with respect to the types of cells which may be included within the capsule membranes. Specifically, it is contemplated that cultures of cells from the tissue of all higher animal~ as well as micro-organisms may be employed. Fused cells, e.g., hybridoma cells, or genetically modified cells produced, for example, by the emerging recombinant DNA technology, can likewise be encapsulated without difficulty. In short, provided there exists a culture medium operable to maintain in vitro the cell type in question, that cell type can be utilized in accordance with the techniques 1~ 7~tj~

1 disclosed herein. Non-limiting examples of the types of substan-ces that may be produced in accordance with the proceæs and by the system of the invention include insulin, glycogen, prolactin, 60matostatin, thyroxin, steroid hormones, pituitary hormones, interferons, folliclegtimulating hormones (FSH), PTH, and antibo-dies .

The system of the invention comprises encapsulated viable cells suspended in an aqueous culture medium. The encap-sulated cells comprise membranes characterized by an upper limit of permeability sufficient to allow traverse of the nutrients needed for cell metabolism and ongoing viability. The membranes enclose viable cells dispo6ed in a medium which includes all components needed to maintain metabolism of the cells and which are of a size range in excess of the upper permeability limit of the membrane. The culture medium comprises components needed to maintain viability of the cells which have a molecular weight below the upper permeability limit of the membranes.

Accordingly, an object of the invention i0 the provi-sion of a system and method for producing biological materials of the type produced by cells. Another object of the invention is to provide such a system wherein the producing cells are con-tained within a protective, healthful microenvironment confined by a semipermeable membrane which serves to separate products of cell metabolism from high molecular weight materials needed for viability and maintenance of the cells. Another object is to provide an improved process for producing biologically active materials from cell cultures. Yet another object i8 to produce antibodies and biological response modifiers such as hormones, interferons, and lymphokines in a serum-free medium.

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1 These and other objects and features of the invention will be apparent from the following description and from the drawing wherein Figure 1 is a schematic diagram illustrating the concept of the invention and Figure 2 is a graph ~howing the results of the experiment de6cribed in example 5.

~ ~L7;~

1 Descri~tion The broad concept of the invention is to interpose a semipermeable membrane about individual cells or groups of cells so as to provide a microenvironment for the cells complete with the cell culture medium and separated by the membrane from an external aqueous medium. Cell6 of mammalian origin typically require for ongoing health and viability the presence of serum proteins, a portion of which have a molecular weight in excess of about 65,000-150,000 daltons. In the prior art technique of un-encapsulated cell culturing, materials of interest secreted from the cells are dispersed in the culture medium and mixed with both high and low molecular weight components. Since the quantities of cell-produced products are typically rather small, isolation of the substance of interest becomes an arduous purification task. Furthermore, mammalian cell cultures are notoriously sen-sitive to contamination by bacterial or other sources. This necessitates that culturing be conducted using various techniques to maintain sterility and often that antibiotics be included in the medium.
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According to the practice of this invention, the foregoing difficulties are alleviated by encapsulating the cells of the culture within semipermeable membranes having a selected limit of permeability generally no greater than about 200,000 daltons, that is, the mernbrane contains pores which allow substances having a maximum molecular weight at or below the upper permeability limit to traverse the membrane whereas substances of molecular weight above the upper permeability limit are precluded from traversing the mernbrane. This allows one to encapsulate cell3 together with a culture medium containing all 1~7;~61 1 componentg needed for ongoing viability, metab~lism, and even mitosis, and then to suspend the so encapsulated cells in a culture medium which contains lower molecular weight substances consumed by the cells but which need not include the required high molecular weight substances.

Typically, cells from higher organisms do not ingest high molecular weight serum proteins and the like, but rather require them in close proximity for ongoing normal biological responses. Salts, amino acids and other lower molecular weight factors which are ingested or metabolized by the cells pass freely through the membrane and may be replenished as needed by simple change of the culture medium external t~ the capsules.
Secreted products of cell metabolism having a molecular weight below the upper limit of membrane permeability collect in the extracapsular medium, where, because of the absence in the medium of the high molecular weight materials, harvesting and isolation of the metabolic product~ of interest are simplified. Harvesting of products of interest having a molecular weight above the upper permeability limit is also aided in that such products collect within the capsules and are not dispersed in the extracapsular volume.

The concept of the invention, as applied to lower molecular weight cell products, is schematically illustrated in the drawing. As shown, a cell 10 is disposed within a capsule membrane 12 having pores 16. High molecular weight factors 18 are enclosed within membrane 12 and are free to circulate within the confines of the membrane in the medium 14. Components 20 needed by the cell as well as metabolic products 22 including the substance of interest 22' freely circulate in both the 13~7i~61 1 intracapsular and extracapsular medium and traverse the membrane through pores 16. As required on a periodic (or continuous) basis, the extracapsular medium together with all of its components can be separated by aspiration or the like from the capsules themselves and replaced with fresh medium. The collected medium will be substantially free of high molecular weight component~ 18, thus simplifying the harvesting and isolation procedures. Furthermore, the cell 10 remains protected within the intracapsular microenvironment at all times.

In 60me cases, e.g., in order to stimulate production by encapsulated cells of a particular substance of interest, it i8 required to subject the cells to high molecular weight com-ponents having molecular dimensions too large to traverse the membrane. An example is the production of interferon from human fibroblasts, leukocytes, or lymphoblastoid cells which are induced to secrete interferon by treatment with certain viruses or high molecular weight nucleic acids. In such a case, if the upper permeablity limit of the membranes is less than the molecular weight of the inducing factor, the cells must be subjected to interferon induction prior to encap6ulation, or the capsule membranes, after culture of the cells, must be ~ selectively disrupted to allow such high molecular weight ~ can~lJ~
tJ~ materials to be ingested by the cell. Copending~application Serial No. ~ , discloses a method of selectively disrupting certain types of capsule membranes which may be used for these and other purposes without damage to the cells.

The process of the invention depends on one's ability to form semipermeable membranes about cells without simultane-ously adversely affecting their ongoing viability. One suitable encapsulation process is set forth in detail below.

1 Cell Encapsulation The tissue or cells to be encapsulated are suspended in an aqueous medium suitable for maintenance or for supporting the ongoing metabolic processes of the particular tissue or cell type involved. Media suitable for this purpose are available commercially. The average diameter of the material to be encapsulated can vary widely between a few micrometers to several millimeters. However, best results are achieved with capsules of a size in the range of 300-1000 micrometers.
10 Mammalian islets of Langerhans are typically 50 to 200 micro-meters in diameter. Individual cells such as fibroblasts, - leukocytes, lymphblastoids, pancreatic beta cells, alpha cells, delta cells, various ratios thereof, or other tissue units 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 dependent, inter alia, on the availability of required nutrients, oxygen transfer, absence of toxic substances in the medium, and the pH of the medium. ~eretofore, it has not been possible to maintain such living matter in a physiologically compatible environment while simultaneously encapsulating. The problem has been that the conditions required for membrane formation have been lethal or harmful to the tissue, and prior to the invention of Canadian application Serial No. 384,524, now Patent No. 1,145,258, no method of membrane formation which allowed tissue to survive in a healthy state had been forthcoming.

However, it has been discovered that certain water-soluble substances which are physiologically compatible with .~

~3L7~$~1 1 living tissue and can be rendered water-insoluble to form a shape-retaining, coherent mass, can be used to form a "temporary capsule" or protective barrier layer about individual cells or groups of cell~ and that this temporary capsule can be treated to deposit a more permanent semipermeable membrane about the cells without damage to the cells. Such a substance is added, typi-cally at a concentration on the order of a few weight percent, to the tissue culture medium, which also contains cells of the culture, serum components (if required) and optionally, a cellu-' 10 lar substr~te such as collagen or another high molecular weight, water dispersible material which acts as an anchoring substrate.
When using collagen, the concentration should be within the range of about 10 ug/ml to about 1 mg/ml, but preferably on the order of 100-500 ~g/ml.

The solution is then formed into droplets containing tis~ue together with its medium and is immediately rendered water-insoluble and gelled, at least in a surface layer.
Thereafter, the shape-retaining temporary cap~ules are provided with a more permanent membrane which may itself ~ubsequently be 6electively disrupted if it i6 desired to release the tissue without darnage. Where the material used to form the temporary capsules permits, the capsule interior may be reliquified after formation of the permanent membrane. This is done by re-establishing the conditions in the medium at which the material is soluble.

The material used to form the temporary capsules may be any non-toxic, water-soluble material which, by a change in ionic environment or concentration, can be converted to a shape-retaining mass. The material should also contains plural, easily ~7~
1 ionized anionic moieties, e.g., carboxyl groups, which can react by salt formation with polymers containing plural cationic groups. As will be explained below, use of this type of material enables one to deposit a permanent membrane of a selected upper limit of permeability without difficulty in surface layers of the temporary capsule.

The presently preferred materials for forming the tem-porary capsule are acidic, water-soluble, natural or synthetic polysaccharide gums. Such materials are commercially available.
They are typically extracted from vegetable matter and are often used as additives to various foods. Sodium alginate is the presently preferred water-soluble gum. Alginate in the molecular weight range of 150,000~ daltons may be used, but because of its molecular dimensions and viscosity will usually be unable to per-- meate the finally formed capsule membranes. Lower molecular weight alginate, e.g., 50,000-80,000 daltons, is more easily removed from the intracapsular volume by diffusion through a membrane of sufficient porosity and is therefore preferred.

Other useable gums include acidic fractions of guar gum, cara-geenan, pectin, tragacanth gum, or xanthan gum.

These materials comprise glycoside-linked saccharide chains. Their free acid groups are often present in the alkali metal ion form, e.g., sodium form. If a multivalent ion such as calcium or aluminum is exchanged for the alkali metal ion, the water-soluble polysaccharide molecules are "cross-linked" to form a water-insoluble, shape-retaining gel which can be resolublized on removal of the ions by ion exchange or via a sequestering agent. While essentially any multivalent ion which can form a salt with the acidic gum is operable, it is preferred that phy-1~7~61 1 siologically compatible ions, e.g., calcium, be employed This tends to preserve the tis6ue in the living state. Other multiva-lent cations can be used. Magnesium ions are ineffective in gelling sodium alginate.

A typical solution composition comprises equal volumes of a cell culture in its medium and a one or two percent solution of gum in physiological saline. When employing sodium alginate, a 1.2 to 1.~ percent solution has been used with success. If the cells to be encapsulated are of the type which require attachment to an anchoring substrate to undergo mitosis, and if the cells are to be grown within the capsules, then collagen or another high molecular weight water-dispersible protein or polypeptide, either natural or synthetic, may be included in the cell culture, and will be confined within the intracapsular volume of the finally formed capsules. If a polymer having plural cationic groups, e.g., polylysine, i~ employed for this purpose, the cationic groups react with anionic sites on the water-soluble gum to form a sub6tantially water-insoluble matrix intertwined with the gum. Preferred concentrations for such materials are on the order of 100-500 ug/ml of suspension (including gum solution).

In the next step of the encapsulation process, the gum solution containing the tissue iB formed into droplets of a desired size. Thereafter, the droplets are immediately gelled to form shape-retaining masses preferably but not necessarily in spherical or ~pheroidal form. The drop formation~
may be conducted by known methods. An exemplary procedure follows .

A tube containing an aqueous solution of multivalent cation~, e.g., 1.5~ CaC12 solution, is fitted with a stopper 1~7~6~

1 which holds a drop forming apparatus. The appartus 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, e.g., a -f~-f~ ~3,y ~~~ O.01 inch I.D.-Tcflon coated needle pagsing 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, with the syringe full of solution containing the material to be encapsulated, the stepping pump is actuated to incrementally force droplets of solution from the tip of the needle. Each drop is "cut off" by the air stream and falls approximately 2.5 cm into the CaC12 solution where it is immediately 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 alginate/cell suspension to assume the most physically favorable shape; a sphere (maximum volume for minimum surface area). Air within the tube bleeds through an opening in the stopper. This results in "cross-linking" of the gel and in the formation of a high viscosity shape-retaining protective tem-porary capsule containing the suspended tissue and its medium.
The capsules collect in the solution as a separate phase and may be separated by aspiration.

In the next step of the process, a semipermeable membrane is deposited about the surface of the temporary capsules by "cross-linking" surface layers. This may be effected by subjecting the gelled temporary capsules to an aqueous solution of a polymer containing cationic group~ reactive with anionic functionalities in the gel molecules. Polymers containing acid /~`c~ f~

:~' .

1~7~$t;1 1 reactive groups such as free imine or amine groups are preferred.
In this situation, the polysaccharide gum is crosslinked by interaction (salt bond formation) between the carboxyl groups and the arnine or imine groups. Permeability can be controlled within limits by selecting the molecular weight of the cross-linking polymer u6ed and by regulating the concentration of the polymer solution and the duration of exposure. A solution of polymer having a low molecular weight, in a given time period, will penetrate further into the temporary capsules than will a high molecular weight polymer. The degree of 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 the pore size. Broadly, polymers within the molecular weight range of 3,000 to 100,000 dalton6 or greater may be used, depending on the duration of the reaction, the con-centration of the polymer solution, and the degree of permeabil-ity desired. One successful set of reaction conditions, using polylysine of average molecular weight of about 35,000 daltons, involved reaction for two minutes, with stirring, of a physio-logical saline solution containing 0.0167 percent polylysine.
This results in membranes having an upper limit of permeability of about 100,000 daltons. Optimal reaction conditions suitable for controlling permeability in a given system can readily be determined empirically in view of the foregoing guidelines.
Using this method it is possible to set the upper permeability limit of the membranes at a selected level below about 200,000 daltons.

Examples of suitable cross-linking polymers include proteins and polypeptides, either natural or synthetic, having ' '7~

1 free amino or imino groups, polyethyleneamines, polyethylene-imines, and polyvinyl amines. Polylysine, in both the D and L
forms, has been used with 6uccess. Proteins such as polyarge-nine, polycitrulline, or polyornithine are also operable.
Polymers in the higher range of positive charge density, e.g., polyvinylamine, vigorously adhere to the anionic groups of the gel molecules to form Qtable mernbranes, but the membranes are rather difficult to disrupt.

Treatment with a dilute solution of gum will tie up free amino groups on the surfaces of the capsules which otherwise may impart to the capsules a tendency to clump.

At this point in the encapsulation, capsules may be collected which comprise a semipermeable membrane surrounding a gelled solution of gum, cell-type compatible culture medium, cells, and optionally an internal matrix of collagen or another anchorage substrate. Since mass tran6fer should be promoted within the capsules and across the membranes, it is preferred to reliquify the gel to its water-soluble form. This may be done by re-establishing the conditions under which the gum is a liquid, e.g., rernoving the calcium or other multifunctional cations from the interiox gel. The medium in the capsule can be resolubilized simply by immersing the capsules in phosphate buffered saline, which contains alkali metal ions and hydrogen ions. Monovalent ions exchange with the calcium or other multifunctional ions within the gum when the capsules are immersed in the solution with stirring. Sodium citrate 601utions may be used for the same purpose, and serve to sequester the divalent ions.

Cell cultures encapsulated as described above may be suspended in culture media designed specifically to satisfy all 1~7 '~1 1 of the requirements of the particular cell type involved and will continue to undergo normal in vitro metabolism. If the culture requires an environment of high molecular weight components such as ~erum components, these may be omitted from the extracapsular medium. Typically, the components normally ingested by cell6 are of relatively low molecular weight and readily diffuse across the cap6ule membranes into the microenvironment of the cell6 where they permeate the cell membrane. Products of metabolism of the cells which are 6ecreted into the intracapsular medium, if they have a molecular weight below the upper limit of permeability of the capsule membrane, likewise diffu6e thereacross and collect in the extracapsular medium.

The encapsulated cell6 may be cultured under conditions of, e.g., temperature, pH, and ionic environment, identical to conventional cultures. Al60, cell-produced products may be har-vested from the extracap6ular medium or from the cap6ules by con-ventional techniques. However, the culturing technique di6closed herein has the following advantages:

1. The cells of the culture are protected from shearing forces and mechanical damage and from contamination by factors having dimensions in excess of the upper permeability limit of the membranes. This means that handling and sterility re~uirements normally incident to culturing procedures can be somewhat relaxed, since microorganisms cannot reach the encap-sulated cells, and viru~ infected cells need not contaminate other cells.

2. The cap6ules in effect immobilize the cells within an environment in which enclosed high molecular weight materials ., ~7;~
1 are confined, yet lower molecular weight cell nutrients and products are readily removed and introduced. This allows the nutrient broth to be intermittently or continuously collected and supplemented as desired, without disturbing the cells.

3. Substances of interest produced by the cells are more easily recovered. Secreted cell products of molecular dimensions small enough to permeate the capsule membranes collect in the extracapsular medium in admixture with nutrients.
However, high molecular weight serum components and the like are not released into the extracapsular medium, thus simplifying recovery of a cell product of interest. Secreted cell products of molecular dimensions in excess of the upper permeability limit of the membranes collect within the capsules. Of course, cell products not secreted through the cell membrane may also be of intere~t. These may be recovered in relatively concentrated form by isolating the capsules and subsequently selectively disrupting the capsule membranes using, for example, the technique disclosed hereinafter, and if necessary by disrupting the cell membranes.

4. The intracapsular volume provides an environment well suited for cell division. Su~pen~ion cultures have been observed to undergo mitosis within the capsule. Anchorage depen-dent cells which in normal cultures grow in a two-dimensional monolayer multiply to form an array within the capsule. Such cells use the interior surfaces of the membrane as a substrate and/or anchor to the high molecular weight materials set forth above which are disposed within the capsule. This leads to significant increases in cell density as compared with conven-tional cultures. The ongoing viability of such cell clusters is aided by the fact that the surface area to volume ratios of the ~7~

capsules can be quite large, and thus all cells have access to re~uired nutrients, oxygen, etc.

In certain situations it would be advantageous to selectively disrupt the capsule membranes to release the cells without damage. One notable example is in the production of interferon (INF). Cells capable of producing INF must be sub-jected to certain viruses or nucleic acids in preparation for the INF production ~tage. Also, in several INF induction procedures, reagents are added to the culture to inhibit protein synthesis.
Accordingly, the growth stage of the culturing process must be conducted under conditions quite different from the INF induction stage. If the substances used for INF induction are of a molecu-lar weight in excess of the upper permeability limit of the cap-sule membranes (as will be the case in virus inductions) the induction process cannot be accompliqhed in the encapsulated cell culture. Accordingly, INF producing cells, if grown within the capsule, would have to be released by disruption of the membrane in order to be subjected to the induction proce~.

Disruption of Membrane~
Cells confined in membranes of the type set forth above rnay be released by a process involving cornmercially available reagent~ having propertie6 which do not significantly adversely affect the encapsulated cell~. First, the capsules are separated from their suspending mediurn, washed thoroughly to remove any ; contaminants present on the exterior of the microcapsules, and then dispersed, with agitation, in a mixed solution of monatomic, multivalent cations such as calcium ions and a stripping polymer having plural anionic moieties such as a salt of a polysulfonic or polyphosphoric acid. Heparin, a natural sulfonated poly-:1~7~

1 saccharide, is preferred for this step. The anionic charge den-sity of the stripping polymer used should be equal to or preferably greater than the charge density of the polyanionic material originally employed to form the membranes. The molecu-lar weight of the polymer should be at least comparable to and preferably greater than the molecular weight of the polymer having plural cationic groups used in forming the membrane.
Within the suspension of capsules in the mixed solution, the calcium ions compete with the polycationic polymer chains used to form the membrane for anionic sites on ~he water-soluble gum.
Simultaneously, the heparin or other polymer having plural anionic moieties dissolved in the 601ution competes with the gum in the membrane for cationic sites on the polymer chains. This result6 in a water-dispersable or preferrably water-soluble com-plex of e.g., polylysine and heparin, and in association of the monatomic cations with molecules of the gel.

This step renders the membrane suseptible to dissolu-tion upon subsequent exposure to a sequestering agent which completes the disruption process by taking up monatomic ions from ; 20 the gel. Capsule membrane debris which remains in the medium, if any, can be easily separated from the cells.

The currently preferred solution for the first stage of the selective disruption process comprises 1.1~ calcium chloride (w/v) and between 500 to 1,500 unit~ of heparin per milliliter of solution. A volume of microcapsules is added to this solution sufficient to constitute between about 20% and 30~ of the total volume of suspension. Calcium chloride and heparin are preferred for disrupting membranes of cell-containing capsules since both reagents are physiologically compatible with most cells and 1~7 ~6i 1 therefore minimize 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 polyphosphoric acid 6alts of the type 6et forth above.

In general, the concentrations of monatomic ions and anionic polymer used in this step may vary widely. Optimum con-centrations may be readily determined empirically, and depend on exposure time as well as the particular polymer used to form the membranes .

The currently preferred sequestering agent for per-forming the selective disruption is sodium citrate, although other alkali metal citrate ealts and alkali metal EDTA salts may also be used. When sodium citrate is employed, the optimum con-centration is on the order of 55 mM. It i8 preferred to di6solve the citrate or other sequestering agent in isotonic saline so as to minimize cell damage.
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The invention will be further understood from the following non-limiting examples.

Example 1: Insulin Production Islets of Langerhans are obtained from human cadaver of animal pancreas and added to a complete tissue culture (CMRL-1969 Connaught Laboratories, Toronto, Canada) at a concentration of approximately 103 islets per milliliter. The tissue culture con-tains all nutrients needed for continued viability of the islets a~ well as the amino acids employed by the Beta cells for making insulin. Four-tenths of a milliliter of a 10-~ islet per milli-- liter suspen~ion is then added to a one-half milliliter volume of 1.2 percent sodium alginate (Sigma Chemical Company) in physiological saline.

:~P~ 61 1 Next, a 1.5 percent calcium chloride solution is used to gel droplets of the solution formed as set forth above.
Droplets on the order of 300-400 microns in diameter emanating from the tip of the needle immediately gel upon entering the calcium solution. The gelled capsules are then transferred to a beaker containing 15 ml of a solution comprising 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% CaC12. After a 3 minute immersion, the capsules are washed twice in 1~ CaC12.

The capsules are then transferred to a 32 ml solution coraprising 1/80 of one percent polylysine (average MW 35,000 daltons) in physiological saline. After 3 minutes, the poly-lysine solution is decanted. The capsules are washed with 1%
CaC12, and optionally resuspended for 3 minutes in a solution of polyethyleneimine (MW 40,000-60,000) produced by diluting a stock 3.3% polyethyleneimine solution in morpholino propane sulfonic acid buffer (0.2M, ph=6) with sufficient lX CaC12 to result in a final polymer concentration of 0.12~. The resulting capsules, having "permanent" semipermeable membranes, are then washed twice with 1% CaC12, twice with physiological saline, and mixed with 10 ml of 0.12 percent alginic acid solution.

The capsules resist clumping, and many can be seen to contain islets of Langerhans. Gel on the interior of the cap-sules i8 reliquified by immersing the capsules in a mixture of saline and citrate buffer (pH-7.4) for 5 minutes. Lastly, the capsules are suspended in CMLR-69 medium.

Vnder the microscope, these capsules are seen to con-sist of a very thin membrane which encircles an islet within 1 7;~
1 which individual cells can be seen. Molecules having a molecular weight up to about one-hundred thousand can traverse the membranes. This allows oxygen, amino acids, nutrient~, and plasma components used in culture media (i.e., lower molecular weight fetal calf plasrna components) to reach the islet an allows insulin to be 6ecreted.

After repeated washings in physiological saline, micro-capsules made in accordance with the above procedure containing approximately 15 iglets are suspended in 3 milliliters of CMRL-1969. When eight days old, in the presence of 600 mg/dl glucose, the capsules secreted into the extracapsular medium, in one run, 67 units/ml insulin in 1.5 ho~rs. In a second run, 68 units/ml insulin were produced in the same amount of time. One week old capsules, in the same medium, but in the presence of 100 mg/dl glucose, in a first run, secreted 25 units/ml insulin in 1.2 hours, and in a second run, secreted 10 units/ml.

^ Exam~le 2: INF-~ Production Human fibroblasts obtained by treating human foreskin tissue with trypsin and EDTA for 5 minutes at 37C in a known ~ C~R~
manner are suspended in a complete growth medium (~Mb~ 1969, Connaught Laboratories~ supplemented with 40% (v/v) purified fetal calf serum, 0.8% sodium alginate (Sigma) and 200 ug/ml purified calf skin collagen. The density of the cell suspension is about 1.5 x 107 cells/ml. Temporary alginate capsules are formed as set forth in Example 1. Semipermeable membranes are deposited in surface layers of the capsules by suspending them in a .005~ (w/v) aqueous solution of polylysine, (MW 43,000 daltons) for 3 minutes.

1~7~
"~ c r: ~ ~
~ The resultinq capsules are suspended in eM~-1969 supplemented with 10~ fetal calf serum. The foregoing steps are all conducted at 37C. After incubation at the same temperature, the capsules, if examined under the microscope, will be found to contain fibroblasts which have undergone mitosis and display three-dimensional fibroblastic morphology within the microcap-sules .

After 4-5 days of incubation, the encapsulated fibroblasts are subjected to an INF-p superinduction techni~ue according to the Vilcek procedure. Under a 5~ CO2 atmosphere (95~ air), the capsule suspension is incubated at 37C for one hour in the presence of lOO ug/ml Poly I-Poly C, a double stranded RNA (known INF-~ inducer) available from PL
Biochemicals, Milwaukee, Wiscon~in and 50 ~g/ml cycloheximide (protein synthesis inhibitor, Calbiochem, La Jolla, California.) After one hour, the suspended capsules are washed in medium ~ RI
(CMLR 1969) containing 50 ,ug/ml cycloheximide and then resuspended in the same solution for 3 hours at 37C under a 5%
C2 atmosphere. At the completion of this incubation the washing step is repeated and the capsules are resuspended in medium containing 50 ug/ml cycloheximide and 5 ug/ml actimomycin D (a known RNA synthesis inhibitor, Calbiochem) and incubated for 2 hours at 37C under a 5~ CO2 a~nosphere. The capsules are then washed twice in medium and suspended in serum-free medium at 37~C
for 18-24 hours, during which time the fibroblasts secrete INF-~, which has a molecular weight on the order of 21,000 daltons and may be harvested from the extracapsular medium.

Example 3: INF-p Production The procedure of Example 2 i~ repeated, except that ?c~ k.

1 ~7~1 1 prior to induction the capsule membranes are selectively disrupted so that the Poly I-Poly C can more ea~ily gain access to the fibroblasts. The disruption procedure is conducted as follows.

10 ml portions of microcapsule suspensions containing about 500-1000 capsules per ml are allowed to settle and the suspen6ion medium is aspirated off. The capsules are washed twice with phosphate buffered saline (PBS, pH=7.4). The washed - capsules are then mixed with a 3.0 ml aliquot of PBS containing 1000 units/ml heparin and 1.1% (w/v) CaC12. The suspension is agitated at 37C for 3 minutes, after which the capsules are allowed to ~ettle, the supernatant is aspirated off, and the cap-; ~ules 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 0.15M NaCl (pH=7.4). The mixture is hand vortexed for 1 minute to induce dissolution of the membranes after which cells are washed twice in medium.

The cells are then suspended in medium, ~ubjected to the induction procedure set forth in example 2, and then re-encapsulated as set forth in Example 2. The capsule suspension is then incubated in serum-free medium for 18-24 hours, during which time INF-~ is secreted from the cells, permeates the cap-sule membranes, and collects in the extracapsular medium.

Examples 2 and 3, if conducted with Poly I-Poly C (5S) (sedimentation value, Poly I and Poly C annealed to form double ætranded RNA) result in the following INF-~ production levels, in units of INF-~/105 cells:

~L7;~

1 Example 2 1. 2.
; 25 25 Example 3 2,500 2,500 Examples 2 and 3, if conducted with Poly I-Poly C (12S) (sedimentation value, double stranded as purchased) result in the following INF-P production levels, in units of INF-~ /105 cells:

Example 2 1. 2.

Example 3 2,500 2,500 The one-hundred fold increase in production using the procedure of Example 3 over that of Example 2 is believed to be due, at least in part, to the fact that the Poly I-Poly C has better access to the cells in the Example 3 procedure.

Exam~le 4:
The procedure of Example 2 is repeated except that cap-sules containing no collagen are employed. The encapsulated cells were grown in conventional monolayer culture, treated with trypin, and induced with Poly I-Poly C (5S) and microencapsulated simultaneously. The extracap6ular medium is found to contain 2,500 units INF-~/105 cells.

ExamPle 5: Monoclonal Antibodies Hybridoma cells obtained from Herman Eisen at MIT were culture to a density of 3.0 x 106 cells/ml. These cells had been fused from mouse pleen cells and mouse myeloma cells in a manner now well known in the prior art and constituted an immortal cell line which in culture produced antibodies against dinitrophenyl 1~72~61 bovine serum albumin. Three ml aliquots of the cell suspension were made up by adding 2.1 ml of suspension containing 1.4%
sodium alginate to 0.6 ml fetal calf serum and 0.3 ml physiologi-cal (150mM) saline. Droplets of the 6uspension were immediately gelled in CaCl~ solution and then treated with a 0.016 weight percent solution of poly L lysine. The interior of the resulting capsules was then reliquified by immersion in a solution of one part llOmM sodium citrate and three parts 150mM saline for 6 minutes. The capsules containing hybridoma cells were then suspended in a mixture of RPMI-1640 medium (Gibco) containing 2096 heat inactivated fetal calf serum.

Cell counts of encapsulated and unencapsulated hybri-ds)ma cultures, and the amount of monoclonal antibody produced by both the encapsulated and unencap6ulated cultures were determined periodically. The re6ults are set forth in graphical form in Fig. 2.

Example 6: INF- o~ from Leukocytes 30 ml buffy coats obtained from the American Red Cross were treated with 3.0 ml of 5% EDTA and repeated 10 minute exposures to 0.8396 NH4Cl at 4C to lyse the red cells. A five minute centrifuge (1200 rpm at 4C) between NH4Cl treatments separated debris from the remaining intact leukocytes. The cells were next suspended in MEM (minimum essential medium, ~erum free - Gibco), diluted by a factor of 100, and stained with tryptan blue for 15 minutes. A cell count conducted on a sample showed that about 1.3 x 109 leukocytes per 30 ml buffy coat survived.
The cells were then suspended at a density of 1 x 107 cells/ml in medium supplemented with 2% heat inactivated fetal calf serum.

11 ~7~

l Induction was effected by exposing the cell suspension to Sendai virug (various concentrations in heamagglutinating units/ml - Flow Laboratories, Md.) for one hour a~ 37C with stirring. The virus was then separated from the cell by centri-fugation at room temperature and the cells were resuspended in equal volumes of MEM - 4~ heat inactivated fetal calf serum and 1.4~ sodium alginate. Capsules were formed as set forth above and then regugpended in serum-free and seruM-containing media.
There were no 6ignificant differences in the ~uantities of INF

detected in the extracapsular medium of these test samples. INF
production levels were also identical in unencapsulated control cultures. The results of these experiments are set forth below Units Sendai Virus INF Produced (HA Units/ml) Units 107 Cells Example 7: INF ~ from Lymphoblastoids Namalwa cells from the American Type Culture Collection were grown both in conventional culture and within microcapsules in RPMI-1640 medium supplemented with 10% heat inactivated fetal calf serum. Volumes of the cell suspensions were then subjected to INF induction and production procedures, with one volume encapsulated and the other unencapsulated. The cultures con-tained substantially equal numbers of cells. To both the encap-sulated and unencapsulated cultures wa~ added 25 mg/ml bromo deoxyuridine in double distilled water to inhibit mitosis. After incubation for 36 hours at 37C, the cells of both cultures were wa~hed and then suspended in RPMI1640 medium supplemented with 2%
heat inactivated fetal calf serum.

1 The encap6ulated culture was then treated to selec-tively disrupt the capsule membranes. The capsules were washed three times in phsiological saline incubated in 1000 units/ml heparin solution containing 1.1% CaC12 for 10 minutes at 37C, and then rewashed in saline. The washed capsules are next incu-bated for 5 minutes at 37C with dilute sodium citrate solution in physiological saline. Agitation of the cap6ule suspension at this point results in dissolution of the membranes and release of the Namalwa cells. The cell suspension is then centrifuged to remove debris and washed several times in citrate/saline solu-tion.

Both cultures were next suspended in fresh RPMI-1640 culture medium supplemented with 2% heat inactivated fetal calf serum and buffer (pH = 7.4) at a density of 1.0 x 106 cells/ml.

To both the conventional culture and the formerly encapsulated culture were then added the Bankowski strain of Newcastle Disease Virus in amniotic fluid. The virus was at a concentration of 1.0 x 108 pfu/ml and was purchased from Poultry Health Laboratories, Davis, California. One ml of the virus was added for each 10 ml of cell suspension. The cultures were incu-bated for 24 hours at 37C.

The conventional culture was then divided into five parts ~1-5 below); the formerly encapsulated culture was divided into 4 partq (6-9 below). Each of the 9 aliquots of culture were then assayed for INF production following the treatments set forth below.

1. untreated 2. resuspended in RPMI-1640 medium with 2%
- heat inactivated fetal calf serum 13~7~

1 3. resuspended in RPMI-1640 medium serum-free 4. encapsulated together with RPMI-1640 medium and

5% heat-inactivated fetal calf serum-capsules suspended in serum-free medium 5. encapsulated together with RPMI-1640 medium and 5% heat-inactivated fetal calf serum-capsules suspended in medium with 2% fetal calf serum

6. resuspended in serum-free mediwn

7. resuspended in medium containing 2% heat-1~ inactivated fetal calf serum

8. reencapsulated together with medium plus 5%heat-inactivated fetal calf serum-capsules suspended in serurn-free medium

9. reencapsulated together with medium plus 5%
heat-inactivated fetal calf serum-capsules suspended in medium plus 2~ serw~l The following table sets forth the quantity of cells required in each of the cell cultures 1-9 to produce 1 unit of INF ~ :

~7~6~

3 - 8 1000, 360 4 680 9 200, 100 . 5 2000 Other embodiments are within the following claims.

What i8 claimed is:

Claims

1. A process for producing a substance which is produced by living cells, said process comprising the steps of:
A. encapsulating said cells within membranes having a selected upper limit of permeability;
B. suspending said encapsulated cells in an aqueous culture medium;
C. allowing said cells to undergo metabolism in vitro and to secrete said substance; and D. harvesting said substance either from said aqueous medium or from within said membranes.

2. The process of claim 1 wherein said encapsulation step (A) is effected by forming said membrane by reaction between cationic groups on polymer chains and anionic groups on a water-soluble gum to form a water-insoluble salt bonded matrix.

3. The process of claim 1 wherein said encapsulation step (A) is effected by the steps of:
1) suspending the cells in an aqueous medium physio-logically compatible therewith and containing a water-soluble gum having plural anionic moieties;
2) forming the suspension into droplets containing the cells;
3) subjecting the droplets to a solution of multiva-lent, physiologically compatible cations to gel the droplets as discrete, shape-retaining, water-insoluble temporary capsules;
and 4) cross-linking surface layers of said temporary cap-sules to produce semipermeable membranes about said droplets by subjecting them to a polymer comprising plural cationic groups reactive with said anionic moieties.

4. The process of claim 3 comprising the additional step of resolubilizing the gel within the membrane produced in step 4).

5. The process of claim 1 wherein said substance has a molecular weight below said selected upper permeability limit, said process comprising the step of allowing the substance to diffuse through said membranes into said aqueous medium and har-vesting said substance therefrom.

6. The process of claim 1 wherein said cells are encapsulated together with a complete cell culture medium sufficient to maintain said cells and to allow biosynthesis of said substance in vitro.

7. The process of claim 1 or 5 wherein the aqueous medium used in step (B) is a complete cell culture medium suf-ficient to maintain said cells and to allow biosynthesis of said substance in vitro.

8. The process of claim 6 wherein a component having a molecular weight in excess of the upper permeability limit of said membranes is needed by said cells in order to allow in vitro biosynthesis of said substance, said process comprising the addi-tional step of encapsulating said component together with said cells.

9. The process of claim 1 or 5 wherein said cells are mammalian cells.

10. The process of claim 1 or 5 comprising the addi-tional step of allowing said cells to undergo mitosis within said capsule.

11. The process of claim 1 or 5 wherein said cells are cells which have been genetically modified.

12. The process of claim 1 or S wherein during said encapsulation step (A), spheroidal microcapsules having a diameter below about 0.5 mm are produced.

13. The process of claim 1 or 5 wherein the substance harvested in step (E) is selected from the group consisting of insulin, glucagon, prolactin, somatostatin, thyroxin, steroid hormones, pituitary hormones, interferons, FSH, and PTH.

14. The process of claim 1 or 5 wherein the substance harvested in step (E) is selected from the group consisting of hormones, interferons, lymphokines, and antibodies.

15. The process of claim 1 or 5 wherein the cells encapsulated in step (A) require contact with a component having a molecular weight in excess of the upper permeability limit of said membranes in order to maintain production of said substance, said component being encapsulated together with said cells in step (A) and the aqueous culture medium used in step (B) being substantially free of said component.

16. The process of claim 1 wherein said selected upper limit of permeability is below about 1.5 x 105 daltons.

17. The process of claim 1 wherein said cells comprise hybridoma cells, said substance comprise monoclonal antibodies having a molecular weight in excess of said selected upper limit of permeability, and said antibodies are harvested from within said membranes.

18. A system for culturing living cells comprising encapsulated viable cells suspended in an aqueous culture medium, said encapsulated viable cells comprising membranes characterized by an upper limit of permeability sufficient to allow traverse of nutrients required by said cells, said membranes enclosing suspended viable cells disposed in a medium which includes all components (A) needed to maintain viability of said cells which are of a size in excess of the upper permeabil-ity limit of said membranes, said aqueous culture medium comprising all components (B) needed to maintain viability of said cells having a molecular weight below the upper permeability limit of said membranes.

19. The system of claim 18 wherein said components (A) comprise serum components.

20. The system of claim 18 wherein said cells comprise mammalian cells.

21. The system of claim 18 wherein said cells comprise microorganisms.

22. The system of claim 18 wherein said cells comprise genetically modified cells.

23. The system of claim 18 wherein said cells comprise hybridoma cells.

24. The system of claim 18 wherein said cells comprise cells capable of secreting in vitro a substance selected from the group consisting of hormones, interferons, lymphokines/ and anti-bodies.

25. The system of claim 18 wherein said cells comprise cells capable of secreting in vitro a substance selected from the group consisting of insulin, glycogen, growth hormones, pituitary hormones, steroid hormones, prolactin, somatostatin, PTH, and FSH.

26. The system of claim 18 wherein said membranes comprise a polymer gum having plural anionic moieties salt bonded to a polymer having plural cationic moities to form a porous matrix.