JPS6244919B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing biological substances of the type produced by cells.

Advances in cell biology have shown that cells of various higher organisms produce small amounts of substances that have significant potential or apparent utility in the treatment or diagnosis of disease. Examples of such substances are well documented in the literature and include various biological response modifiers such as hormones, interferons and lymphokines, as well as other substances such as antibodies for which diagnostic tests are used. Additionally, cell cultures of microbial strains have long been used to mass produce antibiotics.

Particularly in cell cultures from higher animals, there is always a risk of contamination with bacteria and other contaminants. Also, in most cases, the amount of interesting substances produced by culturing cells is very small, and it collects in the culture medium, which contains a complex mixture of serum proteins and other substances. This makes isolation or purification of interesting substances difficult.

Generally speaking, the present invention provides systems and methods for producing substances produced by living cells. By practicing the present invention, two unique advantages have been achieved: providing a protective environment for the cells being cultured and providing a means of collecting the material of interest in a culture medium that is largely free of contaminating foreign components. Ru. The present invention can be used to separate substances of interest from higher molecular weight serum proteins and other analogs normally required to maintain continued viability and metabolism of producing cells. Alternatively, the present invention can be used to collect substances of interest into a culture medium containing relatively small amounts of low molecular weight nutrients or cellular metabolic products.

The method involves enclosing or encapsulating cells in a membrane that is permeable to nutrients, ions, and other relatively low molecular weight materials necessary for normal metabolic functions or continued viability of the cells. This membrane may or may not be permeable to the substance of interest secreted by the cell, but in any case the molecular weight is generally at some selected level of less than about 2.0 x 10 ⁵ Daltons. It has a permeability upper limit sufficient to allow passage of molecules. The capsules thus produced are then suspended in a conventional aqueous culture medium and the cells within the capsules are allowed to undergo normal in vitro metabolic processes. Substances secreted by cells with molecular weights smaller than the permeability limit of the membrane pass through the membrane and collect in the culture medium. Advantageously, high molecular weight substances, such as serum proteins, which are not necessary for the viability or health of various cell cultures from higher animals and are not typically consumed as such, are microencapsulated and confined therein. It is possible to prevent substances from diffusing into the culture medium. When a substance that the cell culture consumes during metabolism has a molecular weight low enough to be able to diffuse through the capsule membrane, it leaves the culture medium and penetrates the membrane.
Also, the metabolic products of the cell, which have molecular dimensions small enough to allow passage through the membrane, diffuse into the culture medium outside the capsule. An interesting substance is
If it has a molecular weight below the permeability limit, it will diffuse into the extracapsular culture medium and can be harvested relatively easily in that culture medium. This is because there are no contaminating high molecular weight materials present in conventional non-encapsulated cell cultures. When the substance of interest has a molecular weight greater than the permeability limit of the membrane, it accumulates within the capsule so that it can be separated from the culture medium and destroyed in preparation for its recovery at a later time.

The present invention is essentially unrestricted as to the types of cells that can be encapsulated within the capsule membrane. especially,
It is contemplated that cultures of cells from microorganisms and tissues of all higher animals may be used. fused cells, such as hybridoma cells, or
Genetically modified cells produced by, for example, modern recombinant DNA techniques can also be easily encapsulated. In short, if a culture medium is available that can maintain cells of the type of concern in vitro, those cells can be used according to the methods disclosed herein. The types of substances that can be produced according to the method and system of the invention include insulin, glucogen, prolactin, somatostatin, thyroxine, steroid hormones, pituitary hormones, interferon, follicle-stimulating hormone (FSH), PTH and monoclonal antibodies. However, the present invention is not limited thereto.

The system used in the method of the invention consists of encapsulated viable cells suspended in an aqueous culture medium. The encapsulated cells have membranes characterized by an upper permeability limit of less than about 1.5 x 10 ⁵ Daltons sufficient to permit passage of nutrients necessary for continued viability or metabolism of the cells. The membrane encapsulates viable cells distributed in a culture medium containing all components necessary to maintain cell metabolism but in a size range greater than the permeability limit of the membrane. The culture medium contains components necessary to maintain cell viability that have a molecular weight less than the upper permeability limit of the membrane.

Accordingly, one object of the present invention is to provide systems and methods for producing biological substances of the type produced by cells. It is an object of the present invention to provide such a system to place productive cells in a healthy protective microenvironment and to separate the metabolic products of the cells from the high molecular weight substances necessary for cell viability or maintenance. The objective is to provide a system confined by a semi-permeable membrane that is useful for Another object of the invention is to provide an improved method for producing biologically active substances from cell cultures. Yet another object of the invention is to produce biological response modifiers such as antibody hormones, interferons, lymphokines in serum-free culture media.

These and other objects and features of the invention will become apparent from the following description and the accompanying drawings.

The broad concept of the invention is to interpose a semipermeable membrane around individual cells or groups of cells so that a small environment for the cells, complete with cell culture medium, is separated by the semipermeable membrane from the external aqueous culture medium. That's true. Cells from mammals typically require the presence of serum proteins to maintain their continued viability or health, some of which are about 65,000 to
It has a molecular weight greater than 150,000 Daltons. In conventional non-encapsulated cell culture, the substance of interest secreted by the cells is dispersed in the culture medium and mixed with both high and low molecular weight components. Typically, the quantities of cellular products are quite small, making isolation and purification of the substance of interest a difficult task. Furthermore, mammalian cell cultures are definitely sensitive to contamination by bacteria and the like. Therefore, culturing must be carried out using various techniques to maintain sterile conditions, and antibodies must often be included in the culture medium.

According to the practices of the present invention, the above problem can be solved by
This is generally alleviated by encapsulating the cultured cells in a semipermeable membrane with a selected permeability range not greater than 200,000 Daltons. That is, a semipermeable membrane is a porous membrane that allows a substance whose maximum molecular weight is the same as or smaller than the permeability upper limit to pass through the membrane. Thus, substances with a molecular weight greater than the upper permeability limit cannot pass through the membrane. From this, cells can be encapsulated together with a culture medium containing all the components necessary for continued viability, metabolic activity and even mitosis, and the cells thus encapsulated can then be
It can be suspended in a culture medium that contains low molecular weight substances (but need not contain the required high molecular weight substances) to be consumed by the cells.

Typically, cells from higher organisms do not take up high molecular weight serum proteins and require something similar for continued standard biological responses. Salts, amino acids, and other low molecular weight elements that are taken up or metabolized by the cells pass freely through the membrane and can be replenished as needed by simply changing the extracapsular culture medium. Cell metabolic secretions with molecular weights below the permeability limit of the membrane accumulate in the extracapsular culture medium. The absence of high molecular weight substances in this culture medium simplifies the collection or isolation of metabolites of interest. Obtaining products of interest with molecular weights greater than the permeability limit is also facilitated by the product being inside the capsule and not dispersed in the extracapsular volume.

The inventive concept as applied to low molecular weight cellular products is schematically illustrated in the accompanying drawings. As shown in FIG.
located within. High molecular weight element 18 also membrane 1
2 and freely circulates within the culture medium 14. Metabolic products 22, including cellular components 20 and substances of interest 22', circulate freely in both the intracapsular and extracapsular culture media and cross the membrane through the pores 16. When operation on a periodic (or continuous) basis is required, the extracapsular culture medium, together with all its components, is separated from the capsule itself, such as by absorption, and replaced by fresh culture medium. The collected culture medium is virtually free of high molecular weight components 18, thus simplifying the harvest and isolation procedure. Furthermore, the cells 10 remain protected within the small environment inside the capsule at all times.

In some cases, for example, in order to stimulate the production of a particular substance of interest by an encapsulated cell, it is required to expose the cell to a high molecular weight component whose molecular dimensions are too large to penetrate the membrane. . One example is interferon production from human fibroblasts, leukocytes, or lymphoblasts that are induced to secrete interferon upon treatment with certain viruses or high molecular weight nucleic acids. In such cases, if the permeability limit of the membrane is lower than the molecular weight of the inducing element, cells can be subjected to interferon induction prior to encapsulation or the capsule membrane can be selectively disrupted after cell culture. The high molecular weight material must be taken up by the cells. U.S. Application No. 243,584, filed on the same day, discloses a special method of selectively disrupting the capsule membrane that can be used for this and other purposes without damaging the cells.

The method of the present invention relies on methods that are capable of forming a semipermeable membrane around cells while not adversely affecting their continued viability. One suitable encapsulation method is detailed below.

Encapsulation of Cells The tissue or cells to be encapsulated are suspended in an aqueous culture medium suitable for preservation or for maintaining continued metabolic activity of the particular type of tissue or cell involved. Culture media suitable for this purpose are commercially available. The average diameter of the raw material to be encapsulated can vary widely from a few μm to a few mm. However, the best results are between 300 and 1000
This is achieved with capsules of size in the μm range.
The mammalian Langerhans is typically 50 to
It has a diameter of 200 μm. Individual cells such as fibroblasts, leukocytes, lymphoblasts, pancreatic beta cells, alpha cells, delta cells, mixtures of various ratios of these cells, or other tissue units can be encapsulated as desired. . Microorganisms can also be encapsulated, including those that have been genetically engineered by recombinant DNA methods or other techniques.

The continued viability of such organisms depends, inter alia, on the availability of necessary nutrients, oxygen transfer, the absence of toxic substances in the culture medium and the pH of the culture medium. It has therefore not been previously possible to maintain such organisms in a physiologically compatible environment and encapsulate them at the same time.
The problem was that the requirements for membrane formation were harmful or lethal to the tissue. Thus, no method of membrane formation that would allow tissue to survive in a healthy state had emerged prior to the invention of US Application No. 24600.

However, certain water-soluble substances that are physiologically compatible with living tissue and that can themselves become water-insolubilized to form shape-retaining aggregates,
Used to form a protective barrier layer or "temporary capsule" around individual cells or groups of cells, which in turn deposits a more permanent semipermeable membrane around the cells without damaging them. It has been discovered that it can be treated as desired. Such substances are added at a concentration of several percent by weight relative to the tissue culture medium. This culture medium contains cultured cells and, if necessary, serum components, and optionally contains a cell matrix such as collagen or a water-dispersible high molecular weight substance that acts as a fixed matrix. If collagen is used, its concentration should be in the range of about 10 μg/ml to about 1 mg/ml, but preferably
The amount should be approximately 100 to 500 μg/ml.

The solution containing the tissue together with its culture medium is then formed into droplets, which are immediately water-insolubilized and gelled at least in the surface layer. This shape-retentive primary capsule is then provided with a more permanent membrane. If it is desired to release the tissue intact, the membrane itself can subsequently be selectively destroyed. Depending on the materials used to form the temporary capsule, the interior of the capsule may be reliquefied after this permanent membrane is formed. This is accomplished by establishing conditions in the culture medium that allow the material to dissolve.

The material used to form the temporary capsule can be any water-soluble, non-toxic material that can be converted into a shape-retaining material by changes in the ionic environment or ionic concentration. The material should also contain a plurality of easily ionizable anionic moieties, such as carboxyl groups, that can react with a polymer having a plurality of cationic groups to form a salt. As will be explained later, by using materials of this type, permanent membranes with a selected upper permeability limit can be deposited without difficulty in the surface layer of the temporary capsule.

Presently preferred materials for forming temporary capsules are water soluble natural or synthetic acidic polysaccharide gums, and such materials are commercially available. These are typically extracted from plants and are often used as additives in various foods. Sodium alginate is currently the preferred water-soluble rubber, and alginates in the molecular weight range of 150,000 Daltons and above can be used, however, because of their molecular weight size and viscosity, the salts cannot penetrate the final formed capsule membrane. Alginate with a lower molecular weight than this, e.g. 50,000-80,000 daltons, is more easily removed by diffusion from the internal volume of the capsule through membranes with sufficient porosity, and it is usually not possible to Therefore it is preferable. Other rubbers that can be used include
Included are guar gum, carrageenan, pectin, tragacanth or xanthan gum.

These materials contain glycosidic linked sugar chains. The free acid groups are often present in alkali metal ionic form, such as sodium ionic form. If a multivalent ion such as calcium or aluminum exchanges this alkali metal ion, the water-soluble polysaccharide molecules become "cross-linked" to form a water-insoluble shape-retaining gel, and the gel is ion-exchanged. It can be redissolved when removing ions by or through a sequestering agent. Although essentially any multivalent ion capable of forming a salt with the acidic rubber is agonistic, it is preferred to use physiologically compatible ions such as calcium. This makes it easier to preserve the tissue in a living state. Other polyvalent cations can also be used. However, magnesium ions are not effective in gelling sodium alginate.

A typical solution composition consists of a medium volume of cell culture and a 1 to 2% gum solution in saline. When using sodium alginate,
1.2-1.6 solutions have been successfully used. If the cells to be encapsulated are of a type that requires attachment to a fixed matrix for mitosis, and if the cells are to be grown inside the capsule, they may be dispersed in collagen or water. High molecular weight proteins or polypeptides, natural or synthetic, can be included in the cell culture and ultimately retained in the internal volume of the formed capsule. If a polymer having a plurality of cationic groups, such as polylysine, is used for this purpose, the cationic groups react with the anionic sites of the water-soluble rubber to form a substantially water-insoluble matrix entangled with the rubber. The preferred concentration of such raw materials is about 100 to 500 μg per ml of suspension (including rubber solution).

In the next step, encapsulation, the tissue-containing rubber solution is shaped into droplets of desired size. Thereafter, the droplets immediately gel and preferably retain a spherical or oblate shape, but do not necessarily have this morphology. Droplet formation can be performed by known methods. An example of the procedure is as follows.

A stopper holding the droplet former is fitted into a tube containing an aqueous solution of polyvalent cations, such as a 1.5% CaCl ₂ solution. The device consists of a housing having an upper air intake and an elongated hollow body frictionally fitted therein. Attached to the top of the housing is a 10 c.c. syringe with a stepped pump, for example, by a Teflon-coated 0.01 inch inner diameter needle extending through the housing along its length. The interior of the housing is designed so that the tip of the needle is exposed to a constant laminar air flow that acts as an air knife. In use, a syringe is filled with a solution containing the material to be encapsulated, and a staged pump is activated to force incremental droplets of solution out of the needle tip. The air flow "breaks" the droplets from each other so that they fall approximately 2.5 cm into the CaCl ₂ solution, where they absorb calcium ions and immediately gel. In this case, the distance between the needle tip and the _CaCl2 solution surface is long enough to cause the sodium alginate/cell suspension to assume the most physically advantageous shape, a sphere (maximum volume for minimum surface area). .
Air within the tube escapes through an opening in the stopper. This results in "crosslinking" of the gel,
It also results in the formation of a highly viscous, shape-retentive, protective primary capsule containing the tissue to be suspended and its culture medium. The capsules accumulate in the solution as a separate phase and can be separated by suction.

In the next processing step, a semipermeable membrane is deposited around the surface of this temporary capsule by "crosslinking" the surface layer. This can be accomplished by exposing the gelled temporary capsule to an aqueous solution of a polymer having cationic groups that can react with anionic functional groups in the gel molecules. Polymers having acid-reactive groups such as free imine or amine groups are preferred. In this situation, polysaccharide gums are crosslinked by interaction of carboxyl groups with amine or imine groups (salt bond formation). By selecting the molecular weight of the crosslinkable polymer used and adjusting the concentration and exposure time of the polymer solution, the permeability can be controlled within a range. A solution of a polymer with a low molecular weight penetrates into the temporary capsule interior more than a high molecular weight polymer over a certain period of time. The degree of penetration of the crosslinking agent is a function of the permeability obtained. Generally, the higher the molecular weight and the lower the penetration, the larger the pore size. Depending on the duration of the reaction, the concentration of the polymer solution and the permeability desired, polymers within the broad molecular weight range of 3000 to 100000 Daltons or more can be used. When using polylysine with an average molecular weight of about 35,000 Daltons, one successful combination of reaction conditions is to react with a physiological saline solution containing 0.0167% polylysine for 2 minutes with stirring. This is approximately
Resulting in a membrane with a permeability upper limit of 100,000 Daltons. Optimal reaction conditions for controlling permeability in a given system can be readily determined experimentally in light of the above guidelines. Using this method, the upper permeability limit of the membrane can be set at selected levels below about 200,000 Daltons.

Examples of suitable crosslinking polymers include natural and synthetic proteins and polypeptides having free amino or imino groups, polyethyleneamine, polyethyleneimine and polyvinylamine. Currently, both the D and L forms of polylysine are used successfully. Proteins such as polyargenin, polycitrulline or polyornithine can also be activated. Polymers with higher concentrations of positive charges, such as polyvinylamine, bind strongly to the anionic groups of the gel molecules and form stable films, but these films are quite difficult to break.

Treatment with a dilute solution of rubber binds free amino groups to the surface of the capsules, but without treatment, the capsules tend to aggregate together.

At this point in encapsulation, the capsules can be collected. It consists of a semipermeable membrane surrounding a solution gel of an internal matrix of rubber, cells, a culture medium compatible with the cells, and optionally collagen or another immobilized matrix. Since mass transfer within the capsule and across the membrane is to be facilitated, it is preferred to reliquefy the gel to return it to its water-soluble form. This may be accomplished by re-establishing the conditions under which the rubber is liquid, for example by removing calcium or polyvalent cations from the internal gel. The culture medium within the capsule can also be redissolved by simply immersing the capsule in phosphate buffered saline containing alkali metal ions and hydrogen ions.
Thus, when the capsule is immersed in the stirred solution,
Monovalent ions exchange with calcium or other multivalent ions inside the rubber. Sodium citrate solution can be used for the same purpose and serves as a sequestering agent to deactivate divalent ions.

A cell culture encapsulated as described above can be suspended in a culture medium specifically designed to meet all the requirements of the particular cell type involved, so that the culture continues to exhibit standard in vitro metabolism.
If the culture requires an environment of high molecular weight components such as serum components, this component can be omitted from the extracapsular culture medium. Components normally taken up by cells are typically of relatively low molecular weight, and as they permeate the cell membrane, they readily diffuse across the capsule membrane and into the microenvironment of the cell. Metabolic products of the cells secreted into the intracapsular medium, if they have a molecular weight smaller than the upper permeability limit of the capsule membrane, likewise diffuse across the membrane and accumulate in the extracapsular medium. .

Encapsulated cells can be cultured under the same conditions as conventional cultures, such as temperature, PH, and ionic environment. Although cell products can also be obtained from the extracapsular culture medium or from within the capsule by conventional techniques, the culture techniques disclosed herein have the following advantages. 1 Cells in culture are protected from contamination by elements with dimensions larger than the upper permeability limit of the membrane;
It is also protected against shear forces or mechanical damage. This means that the handling or sterilization requirements normally associated with culture methods can be relaxed somewhat. This is because microorganisms cannot reach the encapsulated cells, and virus-infected cells cannot contaminate other cells.

2. The capsule renders the cells virtually immobile in an environment in which high molecular weight raw materials are retained, yet lower molecular weight cell nutrients and products are easily removed or introduced. This makes it possible to collect and replenish the nutrient fluid culture medium intermittently or continuously as described above, without damaging the cells.

3. Substances of interest produced by cells are more easily recovered. High molecular weight serum components etc. are not released into the culture medium outside the capsule but accumulate inside the capsule. Recovery of cell products of interest is thus simplified. That is, secreted cell products with molecular dimensions larger than the permeability limit of the membrane accumulate within the capsule. Of course, there are also cell products that are not secreted outside the cell membrane that are of interest. These products can be recovered in a relatively concentrated form by isolating the capsule and subsequently selectively disrupting the capsule membrane and, if necessary, disrupting the cell membrane using, for example, the techniques described below.

4 The intracapsular volume provides a well-suited environment for cell division. Suspension cultures have been observed to undergo mitosis inside the capsule. In standard culture, fixed substrate-dependent cells that grow in a two-dimensional monolayer expand to arrange within the capsule. Such cells use the inner surface of the membrane as a matrix and/or are anchored to the aforementioned high molecular weight materials placed inside the capsule. This results in a significant increase in cell density when compared to conventional cultures.
The continued viability of such cell populations is facilitated by the fact that the capsule has a fairly large surface area to volume ratio, thus all the cells have access to the necessary nutrients, oxygen, etc.

In certain situations, it is advantageous to selectively disrupt the capsule membrane in order to release the cells intact. One notable example is in the production of interferon (INF). Cells capable of producing INF are prepared for the INF production process.
It must be applied to some type of virus or nucleic acid that has been prepared. Some methods of inducing INF also add reagents to the culture to inhibit protein synthesis. Therefore, the culture expansion step must be carried out under completely different conditions than the INF induction step. If the substance used to induce INF has a molecular weight greater than the upper permeability limit of the capsule membrane, the induction procedure cannot be carried out in culture of encapsulated cells (as in the case of viral induction). . Therefore, the INF-producing cells must be released by disruption of the membrane in order to subject them to the induction process as they proliferate within the capsule.

Destruction of Membranes Cells trapped within the types of membranes mentioned above are
Release can be achieved by methods using commercially available reagents that have properties that do not significantly adversely affect the encapsulated cells. First, the capsules are separated from their suspension culture medium and thoroughly washed to remove any contaminants present on the outside of the microcapsules, and then the microcapsules are treated with monoatomic polyvalent cations such as calcium ions and polysulfone. Disperse under stirring in a mixed solution with a stripping polymer having multiple anionic moieties, such as an acid salt or polyphosphate. Preferred for this step is the natural sulfonated polysaccharide heparin. The anionic charge concentration of the stripping polymer used should be equal to, or preferably higher than, the charge density of the polyanionic raw material originally used to form the membrane. The molecular weight of the polymer should be at least commensurate with, and preferably greater than, the molecular weight of the polymer having multiple cationic groups used during membrane formation. In the capsule suspension in the above mixed solution, calcium ions compete with the multi-cationic polymer chains used for membrane formation for anion sites on the water-soluble rubber, and at the same time, for cation sites on the polymer chains, calcium ions compete for anion sites on the water-soluble rubber in the solution. soluble heparin or other polymers with multiple anionic moieties compete with the rubber in the membrane. This results in water-dispersible, preferably water-soluble, complexes of polylysine and heparin, for example, bound to monoatomic cations by molecules of the gel.

This step makes the membrane more susceptible to dissolution when exposed to subsequent sequestering agents. In addition,
The sequestering agent completes the destruction process by absorbing monatomic ions from the gel.
If capsule membrane debris remains in the culture medium, it can be easily separated from the cells.

A currently preferred solution for the first step of selective destruction contains 1.1% (w/v) calcium chloride and 500-1500 units of heparin per ml of solution. One volume of microcapsules is added to enough of this solution to account for about 20-30% of the total volume of the suspension. Calcium chloride and heparin are preferred to disrupt the capsule membrane that encases the cells. This is because both reagents are physiologically compatible with most cells, thus minimizing the potential for cell damage. Aluminum salt or other polyvalent cation (however, Mg〓
Mixtures of (excluding ions) can also be used together with polysulfonates or polyphosphates of the type mentioned.

Generally, the concentrations of monatomic ions and anionic polymers used in this step can vary over a wide range. Optimal concentrations can be easily determined by experiment. As such, it depends on the particular polymer used to form the film and the exposure time.

The currently preferred sequestering agent for selective destruction is sodium citrate, but other alkali metal salts of citric acid and EDTA can also be used. When using sodium citrate, its optimal concentration is around 55mM. Preferably, the citrate or other sequestering agent is dissolved in isotonic saline to minimize cell damage.

Further, the invention will be understood from the following non-limiting examples.

Example 1: Production of Insulin Islets of Langerhans are obtained from human cadavers or animal pancreas and placed in complete tissue culture (CMRL-1969, Connaught Laboratories, Toronto, Canada) at a concentration of approximately 10 ³ islets per ml. Add. This tissue culture contains all the nutrients necessary for the continued viability of the islets, as well as the amino acids used by the beta cells for insulin production.
Then, 0.4 ml of the islet suspension at a concentration of 10 ³ islets per ml is added to a 0.5 ml volume of 1.2% sodium alginate (Sigma Chemical Company) in physiological saline.

A 1.5% calcium chloride solution is then used to gel the solution droplets formed as described above. The droplet, which is approximately 300 to 400 microns in diameter, emerges from the tip of the needle and immediately gels when it enters this calcium solution. The gelled capsules were then mixed with 0.6%
2% 2-(cyclohexylamino)ethanesulfonic acid buffer solution in NaCl (isotonic, PH=8.2)
15 ml of a solution prepared by diluting 1 part with 20 parts of 1% CaCl ₂
Transfer to a beaker containing . After soaking for 3 minutes,
Wash the capsules twice with 1% _CaCl2 .

The capsules were then soaked in 1% polylysine (average molecular weight 35,000 Daltons) 1/80 in physiological saline.
Transfer to 32 ml of solution. After 3 minutes, decant the polylysine solution. 1% capsule
Wash with _CaCl2 and optionally add a 3.3% polyethyleneimine stock solution (0.2M, PH=6) in morpholinopropanesulfonic acid buffer with enough 1% _CaCl2 to give a final polymer concentration of 0.12%. Polyethyleneimine made by diluting (MW40000-60000)
Resuspend in solution for 3 minutes. Obtained "permanent"
The capsule with semipermeable membrane was then washed twice with 1% _CaCl2 , twice with saline, and 0.12%
Mix with 10 ml of alginate solution.

The capsules resist agglomeration and many can be seen to contain islets of Langerhans. The gel inside the capsule is reliquefied by immersing the capsule in a mixture of saline and citrate buffer (PH 7.4) for 5 minutes. Finally, add the capsule to CMLR-69
Suspend in culture medium.

Under a microscope, these capsules can be seen to consist of a very thin membrane surrounding islands within which individual cells can be seen. Molecules with molecular weights up to about 100,000 can cross the membrane. From this, plasma components used in the culture medium (ie low molecular weight fetal bovine plasma components), nutrients, amino acids and oxygen reach the islets and cause them to secrete insulin.

After repeated washing in physiological saline, microcapsules with approximately 15 islets prepared according to the above procedure are suspended in 3 ml of CMRL-1969. 600mg/
After 8 days in the presence of glucose at dl concentration,
Capsules were placed in extracapsular culture medium in one experiment.
67 units/ml of insulin was secreted during 1.5 hours. In the second experiment, 68 units/ml within the same time period.
produced insulin. In addition, in the same culture medium, glucose was present at a concentration of 100 mg/dl.
Week-old capsules secreted 25 units/ml of insulin for 1.2 hours in the first experiment and 10 units/ml in the second experiment.

Example 2: Production of INF-β Human foreskin tissue was treated with trypsin using a known method.
Fibroblasts obtained by treatment with EDTA for 5 minutes at 37°C were treated with 40% (v/v) purified fetal bovine serum,
0.8% sodium alginate (Sigma) and
Complete culture medium supplemented with 200 μg/ml purified calf skin collagen (CMLR-1969 Connaught
Laboratories). The density of the cell suspension is approximately 1.5×10 ⁷ cells/ml. Temporary alginate capsules are formed as described in Example 1. Add this to 0.005% of polylysine (MW43000 Daltons)
A semipermeable membrane is deposited on the surface layer of the capsule by suspending in (w/v) aqueous solution for 3 minutes.

The resulting capsules are suspended in CMLR-1969 culture medium supplemented with 10% fetal bovine serum. All the above steps are performed at 37°C. When the capsule is examined under a microscope after incubation at the same temperature, it is found to contain cells undergoing mitosis and exhibiting a three-dimensional fibroblast morphology within the microcapsule.

After incubation for 4-5 days, the fibroblasts within the capsules are subjected to INF-β hyperinduction technique using the Vilcek method. in Milwaukee, Wisconsin under a 5% CO ₂ (95% air) atmosphere.
Double helical RNA obtained from PLBiochemicals
(Known INF-β inducer) Poly I-Poly C100μ
g/ml and cycloheximide (protein synthesis inhibitor, Radziola, Calif.)
Incubate for 1 hour at 37°C in the presence of 50 μg/ml (Calbiochem). After 1 hour, the suspended capsules are washed with culture medium (CMLR-1969) containing 50 μg/ml of cycloheximide and then suspended again in the same solution for 3 hours at 37° C. under a 5% CO ₂ atmosphere. When this incubation is complete, repeat the washing step and add 50 μg/ml of cycloheximide.
and actymomycin D (a known RNA synthesis inhibitor,
Capsules are resuspended in culture medium containing 5 μg/ml (Calbiochem) and incubated for 2 hours at 37° C. in a 5% CO ₂ atmosphere. The capsules were then washed twice with culture medium and incubated in serum-free culture medium for 18-24 h at 37°C.
Suspend for an hour. During this period, fibroblasts are INF-β
secrete. It has a molecular weight of around 21000 Daltons and is obtained from extracapsular culture medium.

Example 3: Production of INF-β The procedure of Example 2 was repeated except that prior to induction,
By selectively destroying the capsule membrane, polyI-polyC
allows easier access to fibroblasts. The destruction process is carried out as follows.

A 10 ml portion of the microcapsule suspension containing approximately 500-1000 capsules per ml is allowed to settle and the suspension medium is removed by aspiration. The capsules are washed twice with phosphate buffered saline (PBS, PH=7.4). The washed capsules were then treated with heparin 1000
Units/ml and mix with a 3.0 ml aliquot containing 1.1% (w/v) CaCl ₂ . After stirring the suspension at 37°C for 3 minutes, the capsules were allowed to settle, and after removing the supernatant liquid by suction, the capsules were mixed with 0.15M
Wash twice with 3.0 ml of NaCl. After absorbing the second wash, the capsule is mixed with 2.0 ml of an equal volume of a mixed solution of 110 mM sodium citrate and 0.15 M NaCl (PH 7.4). The mixture is manually swirled for 1 minute to induce membrane lysis, after which the cells are washed twice with culture medium.

The cells are then suspended in culture medium, subjected to the induction step described in Example 1, and then encapsulated again as described in Example 1. The capsule suspension is then incubated in serum-free culture medium for 18-24 hours, during which time INF-β is secreted from the cells, which penetrates the capsule membrane and accumulates in the extracapsular culture medium.

When Examples 2 and 3 are carried out using poly I-poly C (5S) (precipitation value, poly I and poly C annealed to form a double helical RNA), it produces INF-β as described below. Amount (INF− per 10 ⁵ cells)
β units): 1 2 Example 1 25 25 Example 3 2500 2500 Examples 2 and 3 were converted into poly I-poly C (12S)
(sedimentation value, double helix as purchased), it results in the following INF-β production (INF-β units per ¹⁰ cells): 1 2 Example 2 25 25 Example 3 2500 2500 The 100-fold higher yield when using the procedure of Example 3 than with Example 2 is at least partially based on the fact that poly I-poly C is more accessible to cells when using the procedure of Example 3. It is believed that there is.

Example 4 The procedure of Example 2 is repeated, but with capsules without collagen. Encapsulated cells were grown in conventional monolayer culture, treated with trypin, induced with poly I-poly C (5S), and simultaneously microencapsulated. The extracapsular culture medium was found to contain 2500 units of INF-β per 10 ⁵ cells.

Example 5: Monoclonal Antibodies Hybridoma cells obtained from Hermam Eisen of MIT were cultured at a density of 3.0 x 10 ⁶ cells/ml. The cells were fused from mouse splenocytes and mouse myeloma cells by methods now well known in the art and constituted an immortal cell line that produced antibodies against dinitrophenyl bovine serum albumin in culture. do. A 3 ml aliquot of the cell suspension was prepared by adding 2.1 ml of a suspension containing 1.4% sodium alginate to 0.6 ml of fetal bovine serum and 0.3 ml of physiological saline (150 mM).
A droplet of this suspension was immediately gelled in CaCl ₂ solution and then treated with a 0.016% by weight solution of poly-L-lysine. The interior of the resulting capsule was reliquefied by immersion for 6 minutes in a solution of 1 part 110mM sodium citrate and 3 parts 150mM saline. The capsules containing the hybridoma cells were then placed in RPMI-containing 20% heat-inactivated fetal bovine serum.
1640 culture medium (Gibco).

Cell counts of hybridoma cultures with and without encapsulation and the amount of monoclonal antibodies produced by these encapsulated and unencapsulated cultures were measured periodically. The results are shown in the graph of FIG.

Example 6: INF-α from white blood cells American Red
30ml of buffy membrane obtained from Cross) with 5% EDTA3.0
ml and lysed red blood cells by repeated 10 minute exposure to 0.83% NH ₄ Cl at 4°C. Centrifugation for 5 min (at 4°C) was performed between treatments with NH ₄ Cl.
As a result, debris was separated from the remaining intact white blood cells. Then cells are MEM
(Minimum Essential Culture Medium, Serum Free, Gibco), diluted by a factor of 100 and triptan blue.
Stained for 15 minutes. Cell counts performed on the samples showed that 1.3 x 10 ⁹ leukocytes per 30 ml of buffy coat survived. The cells were then diluted with 2%
1x in culture medium supplemented with heat-inactivated fetal bovine serum.
The cells were suspended at a concentration of 10 ⁷ cells/ml.

The induction step was performed by exposing the cell suspension to Sendai virus (various concentrations of Heem aggregation (HA) units/ml, Flow Laboratories, Maryland) for 1 hour at 37° C. under stirring. Virus was then separated from the cells by centrifugation at room temperature, and the cells were resuspended in equal volumes of MEM-4% heat-inactivated fetal bovine serum and 1.4% sodium alginate. After capsules were formed as described above, they were resuspended in serum-free and serum-containing culture media. There was no significant difference in the amount of INF detected in the extracapsular culture medium of these test samples. The amount of INF produced in the unencapsulated control cultures was also the same. The results of these experiments are shown below: Sendai virus INF production units (HA units/ml) Number of 10 ⁷ cells 600 10 300 20 150 33 75 50 Example 7: INF-α from lymphoblasts American Type Culture Collection (America Namalwa cells from Type Culture Collection) were grown in a conventional culture medium, and after microencapsulation of the same cells,
RPMI-1460 supplemented with 10% heat-inactivated fetal bovine serum
Grown in culture medium. One volume of unencapsulated cell suspension as well as encapsulated cell suspension was subjected to induced production of INF. Culture media contained essentially equal numbers of cells. Unencapsulated and encapsulated cell cultures were inhibited from mitosis by the addition of bromdeoxyuridine at a concentration of 25 mg/ml in double-distilled water. After 36 hours of incubation at 37°C, both cell cultures were washed and then suspended in RPMI-1640 medium supplemented with 2% heat-inactivated fetal bovine serum.

The encapsulated cell culture was then treated to selectively disrupt its capsule membrane. The capsules were washed three times with saline containing 1.1% _CaCl2 .
After incubation for 10 minutes at 37°C in a 1000 units/ml heparin solution, the cells were washed again with saline. The washed capsules were then incubated with dilute sodium citrate solution in saline for 5 minutes at 37°C. Agitation of the capsule suspension at this point resulted in membrane dissolution and release of Namalwa cells. The cell suspension was then centrifuged to remove debris and washed several times with citrate/saline.

Both cultures were then mixed with 2% heat- ^inactivated fetal bovine serum and buffer (PH
7.4) in fresh RPMI-1640 culture medium.

For native cultures and previously encapsulated cultures,
Bankowski strain of New York disease virus in amniotic fluid was added. The virus was at a concentration of 1.0 x ¹⁰⁸ pfu/ml and was purchased from Poultry Health Laboratories, Davis, California. 1 ml of virus was added for every 10 ml of cell suspension. Cultures were incubated at 37°C for 24 hours.

The conventional culture was then divided into five portions (1 to 1 below).
5) and the previously encapsulated culture was divided into four parts (6-9 below). Nine aliquots of the culture were each tested for INF production after the following treatments.

1 Not treated 2 RPMI containing 2% heat-inactivated fetal bovine serum
1640 3 Resuspend in serum-free RPMI-1640 4 Encapsulate with RPMI-1640 culture medium and 5% heat-inactivated fetal bovine serum and suspend the resulting capsules in serum-free culture medium 5 Encapsulate with RPMI-1640 culture medium and 5% heat-inactivated fetal bovine serum, and suspend the resulting capsules in culture medium containing 2% fetal bovine serum 6 Resuspend in serum-free culture medium 7 2 Resuspend in culture medium containing 5% heat-inactivated fetal bovine serum 8 Re-encapsulate with culture medium + 5% heat-inactivated fetal bovine serum and suspend the resulting capsules in serum-free culture medium 9 Culture medium + 5% heat Re-encapsulation with inactivated fetal bovine serum and culture medium +
The amount of cells required to produce INF-α1 units in each of cell cultures 1 to 9 suspended in 2% serum is shown in the following table: 1 30 6 40 2 45 7 40 3 - 8 1000360 4 680 9 200100 52000 Other embodiments are also practiced within the scope of the following claims.

[Brief explanation of the drawing]

FIG. 1 is a diagram illustrating the concept of the invention, and FIG. 2 is a graph illustrating the experimental results described in Example 5. The explanations of the symbols representing the main parts in Fig. 1 are as follows: 10: Cell, 12: Capsule membrane,
14: Culture medium, 16: Pore, 18: High molecular weight element or serum component, 20: Component necessary for cells, 2
2': Substance of interest, 22: Metabolite.

Claims (1)

[Claims] 1. A method for producing a substance produced by a living cell, comprising: A. placing the cell in a membrane having a selected upper permeability limit of less than about 1.5 x 10 ⁵ Daltons; B. suspending the encapsulated cells in an aqueous culture medium; C. allowing the cells to undergo in vitro metabolism and secrete the substance; D. as a result of the selected upper permeability limit, the capsules A method comprising the steps of accumulating the substance in the capsule, E isolating the capsule from the culture medium, F destroying the capsule membrane, and G recovering the substance from the destroyed capsule. 2. A patent in which the encapsulation step (A) is carried out by forming a membrane by reacting the cationic groups on the polymer chain with the anionic groups on the water-soluble rubber to produce a water-insoluble salt-bonding matrix. The method according to claim 1. 3. The encapsulation step (A) comprises: 1 suspending the cells in an aqueous culture medium containing a water-soluble gum that is physiologically compatible with the cells and having multiple anionic moieties; 2 suspending the suspension, forming droplets containing cells; 3 exposing the droplets to a solution of physiologically compatible polyvalent cations to transform the droplets into discrete or discrete, water-insoluble, shape-retentive particles; gelling as a capsule; 4 crosslinking the surface layer of the capsule by exposing the temporary capsule to a polymer having a plurality of cationic groups capable of reacting with the anion moiety to produce a semipermeable membrane around the droplet; A method according to claim 1, which is performed by steps. 4. The method of claim 3, comprising the additional step of redissolving the gel in the film formed in step 4). 5. The method of claim 1, wherein the cells are encapsulated with a complete cell culture medium sufficient to maintain the cells and allow in vitro biosynthesis of the substance. 6. Claim 1, wherein the aqueous culture medium used in step (B) is a complete cell culture medium sufficient to sustain the cells and allow in vitro biosynthesis of the substance.
The method described in section. 7 To allow in vitro biosynthesis of substances,
6. The method of claim 5, wherein the cell requires a component having a molecular weight greater than the upper permeability limit of the membrane, and includes the additional step of encapsulating the component with the cell. 8. The method according to claim 1, wherein the cells are mammalian cells. 9. The method of claim 1, comprising the additional step of causing the cells to undergo mitosis within the capsule. 10. The method according to claim 1, wherein the cells are genetically engineered cells. 11. The method according to claim 1, wherein in the encapsulation step (A), spherical microcapsules having a diameter of about 0.5 mm or less are formed. 12 The substances recovered in step (G) include insulin, glucagon, prolactin, somatostatin, thyroxine, steroid hormones, pituitary hormones, interferon, FSH and PTH.
The method according to claim 1, wherein the method is selected from the group consisting of: 13. The method according to claim 1, wherein the substance obtained in step (G) is selected from the group consisting of hormones, interferons, lymphokines, and antibodies. 14 If the cells to be encapsulated in step (A) require contact with a component having a molecular weight greater than the permeability limit of the membrane in order to sustain production of the substance, and in step (A) this 2. The method of claim 1, wherein a component is encapsulated with the cells and the aqueous culture medium used in step (B) is substantially free of said component. 15. The method of claim 1, wherein the cells consist of hybridoma cells and the substance consists of a monoclonal antibody having a molecular weight greater than the selected permeability limit, and the antibody is obtained from within the membrane.