JPH048034B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to encapsulated living cells, methods for their production and uses thereof.

The use of cells (whether of animal or plant origin) in vitro is receiving renewed attention with recent technological advances. For example, culturing hybridomas in test tubes is now routinely used to produce highly specific monoclonal antibodies. Cancer cell lines are utilized for the formation of such hybridomas in vitro and for the selection and testing of potential gastrogenic and anticancer compounds. Pancreatic cells are also used for the production and derivation of insulin in vitro and in vivo. Furthermore, the industrial use of isolated non-mobilized cells is attracting attention. This is because they can be used as catalysts for biochemical reactions, and these reactions can be used as important tools for synthesis and analytical decisions (Venkatsubramanian,
(See “Immobilized Microbial Cells,” 106 ASC Symposium Series (1979)).

In many cases, direct introduction of foreign cells into a host generates a severe immune response in the host. For example, if hybridoma cells are grown in the ascites of a host, such as a mouse, the mouse must be pretreated to prevent an immune response. When whole insulin cells are injected into humans, a complex immune response emerges. Therefore, measures are required to facilitate the introduction of such cells into a host and to facilitate the manipulation of cells in vitro.

Many methods have been proposed for the encapsulation or entrapment of biologically active substances that are not living cells.

For example, US Patent No. 4251387; No. 4255411; No.
No. 4,257,884 discloses the production of semipermeable membrane microcapsules by interfacial polymerization and their use in immunoassays and chromatography. The substance to be encapsulated and the hydrophilic monomer are emulsified in a hydrophobic continuous phase, the polymerization is initiated by dissolving the second monomer in this continuous phase, and the polymerization is separated between the hydrophilic droplets and the hydrophobic monomer. It is formed only at the interface with the continuous phase, resulting in the formation of a microporous and ill-defined capsule membrane. The affinity of the continuous phase for hydrophilic monomers can be varied by changing the polarity of the continuous phase, creating microcapsules with uniform capsule membranes and predetermined permeability. US Pat. No. 3,780,195 discloses encapsulation of active substances. According to it, a capsule composition is formed by dispersing the active substance and the shell composition in a solvent for the shell composition, which is formed into particles containing the active substance in the dispersed phase, and then a low molecular weight polyglycol is added. The solvent is removed from the shell composition by. This desolvation operation is facilitated by first dispersing the capsule composition in viscous white mineral oil to form discrete particles and then adding the anhydrous polyglycol. Proteins such as ovalbumin and blood albumin are used as the shell material. Other US Patent No.
2889252; No.3691090; No.3714065; No.3516942; No.
No. 3664936; No. 3642978; No. 3137631 discloses encapsulation of drugs. US Pat. No. 3,137,631 discloses a method for improving the stability of the capsule membrane by encapsulating it in a natural substance such as albumin and then treating it with a crosslinking agent such as formaldehyde or glyoxal.

U.S. Patent No. 4187285 describes a method for homogeneously dispersing various drugs to form soft solid microparticles (not capsules).
No. 4147767 (drugs dispersed in serum albumin); No. 4107288 (drugs dispersed in cross-linked serum albumin particles); No. 3937668 (drugs, insecticides) albumin particles containing agents, dyes, etc.); No. 4024233 (micro-aggregated human serum albumin containing tin dispersed); No.
4094965 etc.

Additionally, microencapsulation of various substances including antigens of various microorganisms, such as bacteria and viruses, is disclosed in U.S. Patent Application No. 1, filed concurrently with the present invention.
Listed in 194127. The method of the application is a method in which an active agent is dissolved or dispersed in a solvent, and a membrane wall forming substance is further dissolved in this solvent, that is,
Dispersing the solvent containing the active agent and wall-forming material in a continuous phase processing medium, evaporating a portion of the solvent from the dispersion process to form microcapsules containing the active material in suspension, and finally , a method in which the remainder of the solvent is extracted from the microcapsules.

Many problems arise when applying these many known techniques to living cells. That is, many of the known techniques have conditions that are too harsh for maintaining the survival of living cells. For example, the use of organic solvents, high temperatures, reactive monomers, and crosslinking conditions compromise the survival of living cells. Furthermore, it is not easy to avoid cell dehydration or osmotic damage. An even bigger problem is the need to provide membrane walls for passage of nutrients and excreted products. If cells are used as a source of micromolecules or biological populations, such as antibodies or virions, the membrane wall must be provided with pores of a size that allow the egress of such micromolecules. When cells are encapsulated and injected into a host, they must be the source of the micromolecules or biological population, and the pores must be of the correct diameter to allow the egress of the micromolecules or population. The microencapsulated cells should be of a size that prevents the entry of host molecules or cells that would destroy the microencapsulated cells due to the host's immune system.

Therefore, living cells can be encapsulated under mild conditions that can maintain their vitality.
There is also a need for a method that can form appropriately controlled pores in the capsule membrane wall.

This invention has been made in view of the above circumstances, and an object thereof is to provide encapsulation of living cells.

That is, the present invention provides a capsule containing living cells, which contains an aqueous suspension of living cells in a capsule made of a cross-linked protein wall.

Additionally, the present invention comprises the steps of: (a) dispersing living cells in an aqueous solution of capsule wall-forming proteins; (c) crosslinking said protein with a crosslinking agent that is soluble in said processing medium and substantially insoluble in said aqueous droplets; It is something to do.

The present invention provides a gentle and efficient production of capsules containing living cells, and is based on the discovery of conditions suitable for the formation of controlled porous capsule membrane walls, resulting in Encapsulated cells can be used in various fields such as hybridoma growth, drug derivation, etc.

As mentioned above, the method of the present invention involves three steps: (a) dispersing living cells in an aqueous solution of capsule wall-forming proteins; (b) dispersing living cells in an aqueous, immiscible, cytocompatible continuous processing medium. (c) crosslinking the protein with a crosslinking agent that is soluble in the treatment medium and substantially insoluble in the aqueous droplets; It is something.

The first step in this method is the formation of a protein solution that ultimately forms the capsule membrane wall. Therefore, the membrane wall material must be soluble in water or a substantially aqueous solution and must be capable of being crosslinked to form a capsule. Suitable examples of this water-soluble protein are casein, collagen, gelatin, soybean protein, gluten, albumin, immunoglobulin, or modified products or derivatives thereof. The shell-forming protein is dissolved in the aqueous solvent, preferably using the minimum practical amount thereof. The amount of solvent should not exceed the viscosity that can be handled by the equipment. moreover,
Its viscosity must be such that good dispersion of the cells is obtained. For example, protein is 5 to 95
% by weight, preferably 5 to 25% by weight. Antioxidants, preservatives, and surfactants may be included at this stage.

To this protein-containing shell-forming solution is added a suspension of cells to be encapsulated. These cells are used by being added to a culture medium containing an appropriate nutrient-containing medium, such as salt, reducing agent, antibiotics, serum, buffer, etc., as described below. Culture media for animal cell lines in vitro or for microorganisms are known. The cells are uniformly dispersed in the shell-forming protein-containing solution using gentle methods to prevent disruption. Another method is to first add a shell-forming protein-containing solution to a suitable culture medium;
This intermediate solution is sterilized and a pellet or other suitable form containing living cells is added thereto. These cells are then dispersed into a suspension. The number of cells added is determined by the desired concentration in the microcapsules, but is generally between 10 ³ and 1 ml of solution.
10 ¹² pieces, preferably in the range of 10 ⁴ to 10 ⁸ pieces.

The second step of the method of this invention is to disperse the aqueous phase in the continuous processing medium. This treatment medium must be immiscible with the aqueous phase. An example of the aqueous immiscible phase is mineral or non-mineral oil, provided that the continuous medium is compatible with the living cells. That is, the continuous phase is required not to impair or interfere with cellular metabolism during manufacture. Examples of this aqueous immiscible phase are silicone oil, peanut oil, cottonseed oil, sesame oil, and the like. Surfactants (emulsifiers) may be added to this continuous processing medium to prevent microcapsules from agglomerating and to control the size of microdroplets in the emulsion. This dispersion is colloid milled,
It is obtained by mechanically stirring the continuous phase processing medium by means of a device such as a homogenizer. A simple mechanical stirrer can also be used and is preferred. The reason for this is that sufficiently gentle stirring is required to prevent cell destruction. Emulsions can also be formed by adding droplets of an aqueous solution to the continuous phase processing medium. A preferred example of this dispersion step is to disperse the aqueous solution in sesame oil.

The temperature at which the aqueous solution is formed and the dispersion of this aqueous solution into the processing medium is not particularly limited, but it does affect the size and quality of the microcapsules.
Furthermore, depending on the type of continuous phase processing medium used, this temperature must not be too low. This is because if it is too low, the aqueous solvent and processing medium will solidify or become too thick and viscous, making it practically impossible to handle.
On the other hand, if this temperature is too high, the treatment medium will evaporate and the cells will lose their vitality. The dispersion step should therefore be carried out at a temperature that allows stable operating conditions to be maintained, preferably in the range from 0°C to 40°C, particularly preferably in the range from 25°C to 37°C.

In the second step, there is no limit to the amount of aqueous droplets as long as a stable emulsion can be maintained. However, it is not good if the amount is too large and cross-linking occurs between two different droplets, or on the other hand it is too small and the microcapsules cannot be recovered after treatment. Ideally the ratio of water to treatment medium should range from 0.1 to 99 parts by volume to 100 parts by volume, more preferably from 1 to 50 parts by volume to 100 parts by volume.

A crosslinking agent is then added to the stable emulsion of aqueous droplets in the processing medium. This crosslinking agent requires many requirements regarding functional groups and structure. First, the crosslinking agent must be substantially soluble in the continuous processing medium and insoluble in the aqueous droplets. This condition is extremely important. because,
This is because this serves as a control element to prevent the introduction of cross-linking agents into the aqueous droplets and thus the possibility of extensive cross-linking between the cells or between the cells and the inner wall of the capsule. Water solubility of the crosslinker must be avoided since most cells contain proteins on their surface and these proteins have groups that can be crosslinked with the crosslinker. Since this crosslinker is almost exclusively soluble in the non-aqueous continuous phase, the cross-linking reaction occurs mostly at the interface between the aqueous droplets and the non-aqueous continuous phase, resulting in interfacial cross-linking.

The second condition is that the crosslinking ability of the crosslinking agent occurs at a suitable manufacturing temperature. The third condition is that it is at least difunctional and can be crosslinked in at least two crosslinkable regions on the protein shell material. The fourth condition requires that the crosslinker have groups that readily react with the natural functional groups of the protein under stable emulsion conditions. Examples of such functional groups are hydroxyl group, amino group,
They are a carboxyl group and a thiol group. More preferably, terminal amino groups or protein amino groups such as the ε-group of lysine are utilized.

A preferred example of the crosslinking agent in the present invention is an oil-soluble halogenated diacid that can form an amide bond with a protein amino group. For example, it is a compound represented by XOC-( _CH2 ) _o -COX (where X is a halogen, preferably fluorine, bromine, or chlorine, and n is a general integer of 4 to 12). The most preferred examples are adipoyl chloride or sebacoyl chloride. In general, any hydroxy- or amine-reactive polyfunctional oil-soluble crosslinker can be used. The fifth condition is that the crosslinking agent does not react with the continuous phase processing medium.

The concentration of crosslinking agent in this treatment medium can be adjusted arbitrarily; the lower limit is the concentration at which the capsule membrane wall cannot form a self-supporting force, and the upper limit is the concentration at which the capsule membrane wall becomes too stiff and impermeable due to too much crosslinking. It is concentration. This upper limit also depends on the solubility of the crosslinking agent in the continuous phase processing medium. The concentration of this crosslinking agent is easily determined by those skilled in the art, and is generally 0.001 to 10 mg/
Used in the range of 100ml (processing medium).

The ratio of crosslinker to shell-forming protein also depends on the desired tonicity of the crosslinker, the number of active crosslinking functional groups on the protein used, droplet size, reaction time, and film thickness. Generally this suitable ratio will be from 1 to 1000 moles of crosslinker per mole of protein.

After addition of the crosslinking agent, the resulting suspension is continuously stirred to maintain the droplets in the emulsion until the desired crosslinking is obtained. The reaction temperature conditions for the crosslinking agent are maintained as described above or slightly increased to promote crosslinking. This time is generally from several minutes to several hours, preferably from 5 to 10 minutes to 2 hours, more preferably from 15 minutes to 1 hour.

In many cases, the suspension of microcapsules obtained in the treatment medium can be used directly into the peritoneum of the animal, for example in the injection of encapsulated hybrid mass. This method is particularly preferred if the processing medium does not contain any crosslinking agent at the end of the reaction and if a biocompatible processing medium, such as sesame oil, is utilized for the formation of the capsules. According to this method, microcapsules can be made in situ and the resulting capsule dispersion can be injected into animals.

Alternatively, the capsules may be separated from the treatment medium and washed with an aqueous or non-aqueous cleaning solution. This separation can be carried out by discontinuing stirring and then by decantation or centrifugation. Additionally, the treatment medium may be formed as a layer on the aqueous phase and the bilayer structure may be centrifuged to force the microcapsules from the treatment medium into the aqueous phase. In this case, the aqueous phase is preferably a culture medium that promotes growth of the encapsulated cells.

In the living cell-containing capsule of the present invention produced as described above, living cells suspended in an aqueous medium are accommodated in the capsule made of albumin crosslinked with a crosslinking agent. The cross-linking agent used for cross-linking the capsule wall generally has a toxic effect on living cells, so when it is eluted from the cell wall, it causes fatal defects such as cell death. However, as described above, the crosslinking agent used in the present invention, particularly sebacoyl chloride, is insoluble in water, so according to the present invention, the above fatal problem can be avoided.

In a preferred embodiment of the invention, the starting protein solution is a water-soluble poragen compound (i.e., a pore-forming or pore-generating compound), such as poly(vinyl alcohol), carboxymethylcellulose, poly(vinylpyrrolidone), starch, and most preferably polyhydric glycols. It may also contain glycols such as The presence of this compound protects the cells during encapsulation and causes the formation of many pores in the capsule membrane wall. This poragen compound is thought to be trapped between protein molecules during the crosslinking process and removed when the aqueous solution comes into contact with the capsule, forming many pores in the membrane wall of the capsule. The amount of poragen compound added can vary within a relatively wide range and depends on the number of cells, the desired porosity, the solubility of the poragen, the amount of capsule composition, etc. Generally, the concentration in the aqueous solution is between 1 mg/ml and 1 g/ml, preferably between 200 and 600.
It would be mg/ml. The ratio of poragen to protein is 1:10 to 10:1 (by weight). The polyglycol used in the present invention has a molecular weight of 100 to
10000's. Among these, polyethylene glycols, particularly those having a molecular weight of 4,000 to 6,000 (more preferably those close to this upper limit) are used.

Another preferred embodiment of the present invention is a method of adjusting the pore size of the capsule membrane wall by bringing a capsule membrane wall-degrading enzyme (or degrading enzyme) into contact with the formed capsule. If the membrane wall of the capsule consists primarily of protein, a proteolytic enzyme, such as trypsin, chymotrypsin, etc., is added to the buffered aqueous suspension of the capsule and propagated for a sufficient period of time to enlarge the pores to the desired size. Generally this time is between 1-2 minutes and 1-2 hours, preferably between 1-2 minutes and 30 minutes. This enzymatic digestion can be accomplished by adding an enzyme inhibitor, such as a proteolytic inhibitor, such as soybean trypsin inhibitor, to the capsule dispersion;
Alternatively, it can be stopped by washing the enzyme out of the capsule.

Another advantageous embodiment is that the capsule wall does not consist only of proteins, but in addition contains 0.1 to 80% by weight of other enzymatically degradable substances, such as polysaccharides, keratin, DNA, etc.
This is the case when it is made of substances such as RNA and collagen. After the formation of capsules whose membrane walls are made of proteins and other enzymatically degradable substances, the pore size of the capsules can be enlarged by treatment with enzymes that degrade the other enzymatically degradable substances. If this substance is a polysaccharide, a polysaccharide degrading enzyme is used. cellulose,
When collagen, DNA, RNA, starch, and keratin are added, cellulase, colanase, and DN
The pore size can be enlarged using enzymes such as Aase, RNase, amylase, keratinase, and the like. The treatment time with this degrading enzyme can be adjusted in the same manner as in the case of the proteolytic enzyme described above.

The capsules of the present invention are spherical particles, which may optionally be irregularly shaped. The capsule is
The diameter can be changed from less than 1 μm to several millimeters. For administration with standard injection needles, those with a diameter of 1 μm or less to 250 μm (microcapsules) are preferred.

The pore size of the membrane wall must be at least large enough to allow the inflow and outflow of nutrients necessary to maintain cell growth and vitality, and must be small enough to prevent the cells themselves from being expelled. Ideally, a pore size that allows the discharge of micromolecules (including known immunoglobulins or virions) with a molecular weight of 10,000 to 500,000 is preferred. Since virions usually have a diameter of about 3000 Å or less, the pore size is
Å to 15 μm, more preferably 20 Å to 0.3 μm.

There are no particular limitations on the nature or type of cells that can be encapsulated in the present invention, and they may be from animals, plants, or microorganisms. The cells may also be artificial, such as hybridoma cells. This artificial material is one of the preferred ones for encapsulation. Other cells obtained from cell lines, such as myeloma cells, can also be encapsulated. Additionally, microbial cells such as bacterial cells can also be encapsulated. Among these, the most preferred bacterial cells are those that secrete pharmacologically useful substances and new bacterial species created by recombinant DNA techniques that are encoded by genes from foreign sources such as mammals, donors, etc. It is a bacterium that represents a substance. Examples of bacterial cells expressing this foreign gene are interferon,
These include growth hormones, insulin (which can of course be produced by the pancreatic cells themselves), other hormones, and prohormone-type molecules.

The number of cells per capsule will vary depending on the size of the capsule, but will be about 1 to 1000, preferably about 1 to 100. Growth and regeneration will increase the cell density within the capsule and growth will reach a saturation level. This saturation level also varies depending on cell type and capsule size. These are experimentally
One would know the saturation limit for a particular cell/capsule size.

Although not intended to fall within the scope of the present invention, the present invention can be applied as a kit utilized by a user for encapsulation of desired cells. The kit generally comprises a carrier having one or more compartmentalized containers for accommodating test tubes, vials, glasses, spheres, and the like. The container may further include a first container means comprising a wall-forming protein together with a poragen, other enzymatically degradable substances, nutritional media, surfactants, etc. The wall-forming substance can be present in solution or in lyophilized form. It is conceivable for the second container means to contain a suitable crosslinking agent and for the third container means to contain a water-immiscible treatment medium. Other container means include those containing desired cells, degrading enzymes, other membrane wall-forming substances, and the like. The kit typically contains instructions in the form of catalogs, booklets, brochures, etc. When using this kit,
The user creates a solution of the substance in the first container means to form a dispersion of cells to be encapsulated, adds said solution to the processing medium, and disperses the aqueous droplets to form a stable emulsion. , just add a crosslinking agent to cause membrane wall formation. of course,
Other arbitrary steps may be further added.

The capsules of the invention are particularly suitable for administration of cells to animals, particularly when it is desired to minimize the animal's immune response to the cells. Thus, by injecting a capsule containing hybridoma cells into an animal, the cells can be grown within the animal. Drug administration is also greatly facilitated by injecting animals with capsules containing cells that produce pharmacologically active substances. Recombinant bacteria that produce insulin or produce growth hormone or interferon, providing a fast-acting and continuous source of these active substances, can also be injected into the animal. Encapsulated pancreatic cells can also be injected into diabetic patients to provide a source of insulin. Cells that produce antibodies, enzymes, and other biologically active substances can also be administered.

Of interest here is a method for producing microcapsules or capsules using proteins from animals used as administration targets or hosts. This reduces the problem of immune response. For example, the use of BSA walls facilitates the use of primarily bovine animals. The use of a protein membrane wall consisting of human serum albumin facilitates administration of the capsule to humans.

Administration of capsules can be carried out by local administration, intravenous administration, intraperitoneal injection, intramuscular injection, infusion, perfusion, and the like.

In addition, the capsules of the invention have other uses. For example, they can serve as catalytic substances in place of conventional immobilized microbial cells or enzymes. Furthermore, it is also possible to use decomposing encapsulated microorganisms to release gases such as oxygen, which can be used for analysis and the like by monitoring this with an oxygen electrode.

When microcapsules are used for therapeutic purposes, the dosage is determined in consideration of age, gender, patient condition, other concurrently administered drugs, side effects, and the like. For example, the degree to which insulin and interferon are released from the circulatory system per certain period of time can be easily calculated for a given subject, and thereby an appropriate amount of microcapsules containing a given cell can be injected.

Particularly useful are capsules in which the protein wall-forming material is partially or entirely derived from immunoglobulins. By selecting a particular type of immunoglobulin, one can create a capsule that is targeted to the antigen site with the aid of membrane-containing antibodies, and this capsule can be turned into a designated carrier system for living cells.

(Example) Production example of hybridoma microcapsules After disinfecting all microencapsulation equipment, add bovine serum albumin and BSA (100 mg).
Dissolved in 1 ml of RPM1 1640 culture medium (containing bicarbonate of soda, 2-mercaptoethanol, penicillin, streptomycin, mycozoa and 10% heat-inactivated fetal calf serum). Filter this solution through a Millipore filter (type HA, 0.45 μm, Millipore)
Bedford, Mass., USA) and sterilized by passage into sterile test tubes. Next, polyethylene glycol (PEG,
Carbowax6000 (product name) Fisher Scientific,
Pittsburgh, USA) was dissolved in this BSA solution and a pellet containing about 10 ⁶ hybridoma cells was suspended in 0.5 ml of BSA-PEG media mixture.

This suspension was added dropwise to 20 ml of sterilized sesame oil contained in a 50 ml resin kettle at a temperature of 37° C. and a stirring speed of 1200 rpm. As a result, a water/oil emulsion containing aqueous microdroplets consisting of BSA, PEG, cells and culture medium was obtained. One minute after adding this aqueous phase to sesame oil, sebacoyl chloride (2 ml) was added.
(0.2 ml dissolved in sesame oil) was added to the resin pot. Two minutes after this addition, the stirring speed was increased.
Reduced from 1200rpm to 900rpm. in this state
It was maintained for 40 minutes to crosslink sebacoyl chloride to BSA and form microcapsule walls. 40 more
After several minutes, the resin kettle was gently centrifuged to sediment the microcapsules. After removing the supernatant, the microcapsules were washed with fresh sterile sesame oil to remove residual sebacoyl chloride.

In order to transfer the microcapsules to the culture medium, the microcapsules were centrifuged again and the supernatant liquid was removed. An additional 2 ml of heptane was added to the microcapsule pellets and immediately mixed with 5 ml of culture medium. The mixture was centrifuged, the supernatant was removed, and the microcapsule pellets were washed again with fresh culture medium and adjusted to the appropriate pH.

Cell viability after microencapsulation could be demonstrated by culturing the microcapsules in neutral red. In other words, this microencapsulated cell was shown to have the ability to survive by staining this neutral red. As a second step, the microcapsules were opened and the exposed cells were incubated with trypan blue. As a result, no staining of the cells was observed, indicating the viability of the cells. The cells showed viability for at least 2 months.

Claims (1)

[Scope of Claims] 1. A living cell-containing capsule containing living cells in a capsule made of an albumin wall cross-linked with sebacoyl chloride. 2. The living cell-containing capsule according to claim 1, wherein the albumin wall further contains a water-soluble poragen compound. 3. The living cell-containing capsule according to claim 1, wherein the albumin wall further contains an enzyme-degrading substance. 4. The living cell-containing capsule according to claim 3, wherein the enzymatically degraded substance is polysaccharide, protein, or nucleic acid. 5. The living cell-containing capsule according to claim 1, wherein the albumin wall cross-linked with sebacoyl chloride is porous and can allow passage of cell nutrients. 6 Claim 1 in which the albumin wall cross-linked with sebacoyl chloride is porous and is capable of passing molecules or particles with a molecular weight of 500,000 or less.
Capsules containing living cells as described in Section 2. 7. A capsule containing living cells according to claim 6, wherein the molecule is an immunoglobulin. 8. The capsule containing living cells according to claim 7, wherein the immunoglobulin is a monoclonal antibody. 9. A capsule containing living cells according to claim 6, wherein the molecule is virion. 10. The capsule containing living cells according to claim 1, wherein the albumin wall has a large number of pores with a diameter of 5 angstroms to 15 μm. 11. The capsule containing living cells according to claim 1, wherein the cells are selected from animal, plant, microorganism, and artificial cells. 12. The capsule containing living cells according to claim 11, wherein the cells are hybridoma cells. 13. The capsule containing living cells according to claim 11, wherein the cells are virus-infected cells. 14. The capsule containing living cells according to claim 11, wherein the cells are bacterial cells. 15 The bacterial cell is
15. A capsule containing living cells according to claim 14, which produces a product encoded by a non-bacterial gene recombined into DNA. 16. The capsule containing living cells according to claim 15, wherein the non-bacterial gene is a mammalian gene. 17. A capsule containing living cells according to claim 1, in combination with a nutritional medium for said cells. 18. Capsule containing living cells according to claim 17, wherein the nutrient medium is an in vitro medium. 19. The capsule containing living cells according to claim 17, wherein the nutrient medium is an in-vivo medium. 20. The capsule containing living cells according to claim 17, wherein the in-vivo medium is animal ascites. 21. The capsule containing living cells according to claim 1, wherein the capsule has an average diameter of 250 μm or less. 22 (a) dispersing living cells in an aqueous solution of albumin forming the capsule wall; (b) dispersing the cell-containing medium in aqueous droplets in a water-immiscible and cytocompatible continuous processing medium; (c) crosslinking said albumin with sebacoyl chloride; 23. The method of claim 22, wherein the aqueous solution of albumin contains nutrients for the cells. 24. The method of claim 22, wherein the solution further comprises an enzyme-degrading substance. 25. The method according to claim 24, wherein the enzymatically degraded substance is polysaccharide, protein, or nucleic acid. 26. The method according to claim 22, wherein said cells are selected from animal, plant, microorganism, and artificial cells. 27. The method of claim 26, wherein the cells are hybridoma cells. 28. The method of claim 26, wherein the cell is a bacterial cell. 29 The bacterial cell is
29. The method of claim 28 for producing a product encoded by a non-bacterial gene recombined into DNA. 30. The method of claim 29, wherein the non-bacterial gene is a mammalian gene. 31. The method according to claim 22, wherein the capsules have an average diameter of 250 μm or less. 32. The method according to claim 22, wherein the treatment medium is vegetable oil or mineral oil. 33. The method of claim 22, wherein the treatment medium is selected from peanut oil, sesame oil, and cottonseed oil. 34 Claim 22 further comprising the step of washing the crosslinked capsule with a water-insoluble solvent
The method described in section. 35. The method of claim 22, wherein the aqueous solution of albumin forming the capsule wall further comprises a water-soluble poragen compound. 36 The poragen compound is added to the solution from 1 mg to
36. The method of claim 35 comprising 1 g/ml. 37. The method of claim 35, wherein the poragen compound is poly(vinyl alcohol), carboxymethylcellulose, poly(vinylpyrrolidone), starch or glycol. 38 The poragen compound has a molecular weight of 100 to
36. The method of claim 35, wherein the polyglycol is 10,000. 39. The method according to claim 22, comprising the step of adding a capsule wall-degrading enzyme to a capsule wall made of albumin cross-linked with sebacoyl chloride for a sufficient period of time to enlarge the pores in the wall to a predetermined average size. Method. 40. The method of claim 39, wherein the enzyme is a proteolytic enzyme. 41 Claim 24 further comprising the step of adding a capsule wall degrading enzyme to the capsule wall made of albumin crosslinked with sebacoyl chloride for a sufficient period of time to enlarge the pores in the wall to a predetermined average size. Or the method according to any of paragraph 25. 42 The enzyme is polysaccharide degrading enzyme, DNase, RNase, keratinase, cellulase,
42. The method according to claim 41, wherein the method is selected from amylase and collagenase.