JPS6025929A - Encapsulated cell, manufacture and use - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

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

Cells in vitro (whether of animal or plant origin)
The use of is receiving new attention due to recent technological advances. For example, culturing hybrid masses in test tubes is now routinely used for the production of specific monoclonal antibodies. The non-carcinogenic lines are utilized for the formation of such hybrid masses in vitro and for the screening and testing of potentially carcinogenic and bedside cancerous compounds. Pancreatic cells are also used for the production and delivery 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 determination (V@nkatsubr
amanian+ ``immobilized microbial cell l'.

1.06 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 needed 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, a US patent! 4,251.387 ; Genus 4.2
55.411; & 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 formed by dissolving the hydrophilic droplets and the hydrophobic 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 Patent A3,780,195 discloses encapsulation of active substances. According to this, a capsule composition is formed by dispersing the active substance and the shell composition in a solvent for the shell composition, 1 this is made 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. As the shell material, egg and protein such as blood albumin are used. Other U.S. Patent Office 2,889,252
;A3,691,090;Ni3,714,065;3
,516,942;A3,664,93.6;A3,
642,978; A 3.137.631 discloses the encapsulation of drugs. US Patent A 3.137.
No. 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.

A method for homogeneously dispersing various drugs to form soft solid microparticles (not capsules) is disclosed in U.S. Patent A4,18.
7.285 (technetium-9.9m in albumin)
'z Dispersion); Boiled 4, 147.767 (Drug dispersed in serum albumin); Boiled 4,107
, 288 (drugs dispersed in cross-linked serum albumin particles); Boiled 3,937,668 (drugs, insecticides,
Albumin particles containing dye etc.); A4,024
, 233 (micro-aggregated human serum albumin with tin dispersed therein); A4,094,965, etc.

Microencapsulation of a wide variety of substances, including antigens of various microorganisms, such as cutilia, viruses, etc., is also described in co-pending U.S. Patent Application No. 194,127. The method of the application is a method of dissolving or dispersing an active agent in a solvent and further dissolving a film wall-forming substance in this solvent, that is, a method in which a solvent containing an active agent and a film wall-forming substance is dispersed in a continuous shaft processing medium. The method involves evaporating a portion of the solvent from the dispersion step to form microcapsules containing the active substance in suspension, and finally extracting the remainder of the solvent from the microcapsules.

Many problems arise when applying these many known techniques to living cells. In other words, 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 molecules, 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, there has been a need for a method that can encapsulate living cells under mild conditions that can maintain the vitality of the living cells, and that can form appropriately controlled pores in the membrane wall of the Cassicel.

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

That is, the present invention provides a composition containing living cells housed in a case cell made of a crosslinked tongue chamber wall.

Further, the present invention comprises (a) dispersing living cells in an aqueous solution of capsule wall-forming tongue and lining material; forming aqueous droplets of a cell-containing medium; (c) crosslinking the tongue and tongue with a crosslinking agent that is soluble in the treatment medium and substantially insoluble in the aqueous droplets; The present invention provides a method for encapsulating living cells comprising:

The present invention provides a gradual and efficient production of encapsulated 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 a wide range of areas, including hybridoma growth, drug derivation, etc.

As described 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, cell-compatible continuous processing medium; forming aqueous droplets of the cell-containing medium; (a) crosslinking the tongue-matrix with a crosslinking agent soluble in the treatment medium and substantially insoluble in the aqueous droplets; The process is a random thing.

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 substantially aqueous solutions and must be capable of being crosslinked to form capsules. Suitable examples of water-soluble tannock substances include casein, collagen, gelatin, and soybean tannin. 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. Furthermore, the viscosity must be such that good dispersion of the cells is obtained. For example, the amount of tongue and buttock material is 5 to 95% by weight, preferably 5 to 25% by weight. Antioxidants, preservatives, and surfactants may be included at this stage.

This tan/J? Add the suspension of cells to be encapsulated to the shell-forming solution. 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 a gentle method to prevent destruction. Another method is to first add the shell-forming protein-containing solution to a suitable culture medium, sterilize this intermediate solution, and add to it a pellet or other suitable form containing living cells. 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 10 to 10 cells, preferably 10 to 10 cells per solution.
Used in a range of 8.

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. This aqueous immiscible phase must be stripped of mineral oil or non-mineral oil and must be completely compatible with the living cells and this continuous medium during production. It is required that it not harm or interfere with cellular metabolism. Examples of this aqueous immiscible phase are silicone oil, peanut oil, cottonseed oil, sesame oil, and the like. Surfactant (emulsifier)
may be added to this continuous processing medium to completely prevent agglomeration of the microcapsules or to control the size of the microdroplets in the emulsion.

Is this dispersion colloid mill or homo? It is obtained by mechanically agitating the continuous phase processing medium by means of a device such as a nizer. 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 this 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 will affect the size and quality of the microcapsules. Furthermore, depending on the type of continuous phase processing medium used, its 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. This dispersion step is therefore carried out at a temperature that can be maintained under stable operating conditions, preferably I'io.
℃ to 40℃, particularly preferably 25℃ to 3℃
It should be in the range of 7°C.

In the second step, there is no limit to the amount of aqueous droplets as long as a stable emulsion can be maintained. It is not good if the force is too large and cross-linking occurs between two different droplets, or if 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. This is because this constitutes a control element to prevent the introduction of cross-linking agents into the aqueous droplets and thus the possibility of extensive cross-linking between cells or between cells and the inner wall of the capsule. be. Water solubility of the cross-linking agent must be avoided since most cells contain on their surface tunica and these tunica possess groups that can be cross-linked with the cross-linking agent. 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 or more crosslinkable regions on the charcoal material. The fourth condition requires that the crosslinking agent have a group that readily reacts with the natural functional groups of the protein under stable emulsion conditions. Examples of such functional groups are hydroxyl, amino, carboxyl, thiol groups. More preferably, terminal amine groups or protein amine 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 halodanated diacid, which is capable of forming an amide bond with a tan/matrix amino group.

For example, it is a compound represented by is adipoyl chloride or sebacoyl chloride.Generally, hydroxy or amine-
Any reactive multifunctional 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 Cassicell membrane wall cannot form a self-supporting force, and the upper limit is the concentration at which the 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 10/10
It is used in the range of 0 m1 (processing medium).

The ratio of crosslinking agent to shell-forming protein also depends on the desired tonicity of the crosslinking agent, the number of active crosslinking functional groups on the tongue/spear material used, droplet size, reaction time, and film thickness. Generally, a suitable ratio will be from 1 to 1,000 moles of crosslinking agent per mole of tongue/silk.

After addition of the crosslinking agent, the resulting suspension is continuously stirred,
The droplets are maintained in the emulsion until the desired crosslinking is achieved. 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 resulting suspension of microcapsules in a treatment medium can be used directly in the animal's reconvulsion, for example in the injection of encapsulated hybrid masses. This method is particularly preferred if the treatment medium does not contain any crosslinking agent at the end of the reaction and if a treatment medium compatible with organisms, 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 interrupting the agitation and then decantation or centrifugation.The treatment medium is then formed as a layer on the aqueous phase and the structure is centrifuged to release the microcapsules under the force of the treatment medium.・It may be forcibly transferred to the aqueous phase. The aqueous phase in this case is preferably a culture medium that promotes growth of the encapsulated cells. In a preferred embodiment of the invention, the starting tongue solution contains a water-soluble poratan compound (i.e., a pore-forming or promiscuous compound), For example, eyes? (vinyl alcohol), cal (xymethylcellulose), poly(vinylpyrrolidone),
It may also contain starch, most preferably glycols such as polyhydric glycols. The presence of this compound preserves the cells during encapsulation and causes the formation of many pores in the capsule fiJ m. It is believed that this poratan compound is trapped between the tan/4' lithium molecules during the crosslinking process and is removed when the aqueous solution comes into contact with the capsule, forming many pores in the membrane wall of the capsule. The amount of poratan compound added can vary within a relatively wide range and will depend on the number of cells, the desired porosity, the solubility of Polaque 9, the quality of the capsule composition, etc. Generally, its concentration in aqueous solution is lyy/ml or L 19 /rn
l, good tL<1d200-600 would be a school detective. Ratio of Boradan to protein fdl: 10-10:
1 (weight). The raw polyglycol molecule m'' used in the present invention is 100 to 1'0.000.

Among these, Noriethylene glycol, especially molecular 74,0
00 to 6,000 (more preferably close to this upper limit).

Another preferred embodiment of the present invention is a method of adjusting the pore size of the membrane wall of the capsule by bringing the capsule membrane wall degrading enzyme (or degrading enzyme) into total contact with the formed capsule. If the membrane wall of the capsule is primarily composed of tunicin, a proteolytic enzyme, such as trizocin, gymotrigcin, etc., is added to the buffered aqueous suspension of the capsule and allowed to propagate 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 also be stopped by adding enzyme inhibitors, such as tongue decomposition inhibitors, such as soy trinosine inhibitors, to the capsule dispersion or by washing away the enzyme from the capsule force.

Another preferred aspect is power! The membrane wall of the cell is not mainly composed of protein and cephalic substances, and the weight ratio of 0.1 to 80% is other enzymatically degradable substances, such as polysaccharides, keratin,
This is the case when it is made of substances such as DNA, RNA, and colladan. 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 saccharide-degrading enzyme is used. Cellulose 1 collagen, pNA, RN
A1 starch, cellulase if keratin is added,
Pore size can be expanded using colanase, DNase, RNase, amylase, keratinase, etc. The treatment time with this degrading enzyme can be adjusted in the same manner as in the case of the tongue and spear degrading enzyme described above.

The capsules of the present invention are spherical particles, which may optionally be irregularly shaped. Capsules can vary in diameter from less than 1 μm to several millimeters in diameter. For administration with standard injection needles, those with a diameter of 1 μm or less to 250 μm (microcassicles) 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 from expelling themselves. . Ideally the molecular weight is 10,000-50
0. A pore size that allows the evacuation of '000 micromolecules (including known immunoglobulins or gyrions) is preferred. Rielion is usually about 3. Since the diameter is less than '000X, the pore size should be 5x to 15μm, preferably 20X to 0.3μm. There are no particular restrictions on the nature or type of cells that can be encapsulated in the present invention, including those from animals. , from plants, or from microorganisms. The cells may also be artificial, such as N. ipridoma cells. Other cells obtained from the O cell line, which is one of the preferred for this artificial Mononoke encapsulation, such as myeloma
a) 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 cells created by DNA recombination techniques that are encoded by genes from foreign sources such as mammals, donors, etc. It is a bacterium that represents the attached substance. Examples of bacterial cells expressing this foreign gene include interferon, growth mormon, insulin (which can of course be produced by pancreatic cells themselves), other mormons, noromormon m molecules, etc. The number of cells varies from 1 to 100, depending on the size of the capsule.
0. Preferably it will be 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 the cell type and casselle size.

These depend experimentally on knowing the saturation limit for a particular cell/capsule size.

The present invention further provides for the manufacture of a kit for use by a user for encapsulation of desired cells. The kit generally consists of a carrier having one or more compartments for containing test tubes, vials, glasses, spheres, etc. The container may further include a first container means consisting of a membrane-walled tongue material along with poladan, other enzymatically degradable materials, nutritional media, surfactants, and the like. The wall-forming material can be present in solution or freeze-dried. As a second container means,
as a third container means containing a suitable crosslinking agent;
Containing a water-immiscible treatment medium is contemplated.

Other container means include those containing desired cells, degrading enzymes, other membrane wall-forming substances, and the like. This kit usually contains catalogs, pamphlets, instructions in Noyanfret, etc. When using this kit, the user must
forming a solution of the substance in a container means to form a dispersion of cells to be encapsulated, adding said solution to a processing medium and dispersing the aqueous droplets to form a stable emulsion;
Simply 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 the administration of cells to animals, especially when it is desired to minimize the animal's immune response to the cells; therefore, by injecting the cassette containing the hybridoma cells into the animal. , the cells can be grown within the animal. Drug administration is also greatly facilitated by injecting the animal with a cassette containing cells that produce the pharmacologically active substance.

Recombinant bacteria that produce insulin, growth hormone or interferon, and provide an immediate and continuous source of these active substances, can also be injected into the animal. Cassicellated 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 cassels using tongue material 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 bovine animals as a receptacle. The use of a tongue/macro 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, technology can serve as a catalytic material in place of traditional 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 amount of incilin or interferon to be released into the circulatory system per given period of time can be easily calculated for a given subject, and thereby an appropriate amount of microcassicles containing a given cell can be injected.

Particularly useful are casselles in which part or all of the wall-forming material is made from immunoglobulins. By selecting a specific type of immunoglobulin, one can create a capsule that is targeted to the antigen site as an adjunct to membrane-containing antibodies, turning this capsule into a designated carrier system for living cells.

(Example) After disinfecting all microencapsulation equipment, bovine serum albumin and BSA (100 m9) were added to RPMI.
1640 culture medium (soda bicarbonate, 2-Merka 7') containing ethanol, dicarboxylic acid, streptomycin, mycozoa and 10 g heat-inactivated fetal bovine serum) 1 m
It was dissolved in l. This melt O, Millipore filter (Tie 7'HA, 0.45μm, Millipore Bedford)
, Massachusetts.

Sterilized by passage through a sterile test tube (US). Next, polyethylene glycol (PEG, Ca
rbowax 6,000 (Product name) Fisher
Scientific, Bitz Library, USA) 20
0 cells were dissolved in this BSA solution and the pellet containing approximately 10 cells was placed in the BSA-PEG medium mixture.
．． It was suspended in 50ml.

This suspension was mixed with 20% sterilized sesame oil in a 50% resin pot.
It was added dropwise to m1 at a temperature of 37° C. and a stirring speed of 1.20 rpm. As a result, B.S.A.

A water/oil emulsion containing aqueous microdroplets consisting of PEG, cells and culture medium was obtained. One minute after adding this aqueous phase to the sesame oil, sebacoyl chloride (0.2 d dissolved in 2 ml of sesame oil) was added to the resin kettle. Two minutes after this addition, the stirring speed was increased to 1.20 Or
pm, to 90 Orpm.

decreased to This state was maintained for 40 minutes to crosslink sebacoyl chloride to BSA and form microcapsule walls. After a further 40 minutes, the resin pot was statically centrifuged to sediment the microcapsules. After removing the supernatant, the microcapsules were washed with fresh sesame oil to remove residual sebacoyl chloride.

In order to transfer the microcassicles to a culture medium, the microcapsules were centrifuged again and the supernatant liquid was removed. Further, it was added to the Hegetan 2d'r microcapsule pellet and immediately mixed with 5 ml of culture medium. The mixture was then centrifuged, the supernatant removed, and the microcapsule pellets washed again with fresh culture medium and adjusted to the appropriate pH.

Cell viability after microencapsulation could be demonstrated by culturing the microcassettes in neutral red. In other words, this microencapsulated cell stained this neutral red color and was shown to have vitality. As a second step, the microcapsules were opened and the exposed cells were cultured with Tri-N-Blue. As a result, no staining of the cells was observed, indicating the vitality of the cells. The cells showed viability for at least 2 months.

Applicant's representative Patent attorney Takehiko Suzue■, Indication of the case Patent application No. 1982-131097 2, Name of the invention Encapsulated cells, manufacturing method and uses thereof 3 Person making the amendment Relationship with the case 111 people filed the patent application Thestor Research and Dayperots Dement Corporation 4 Agent Address: 1-chome Toranomon, Minato-ku, Tokyo [126 City 55 Daiichi Mori Building 105 li 03 (502) 3181 (
Main representative) 37. Contents of the amendment: Reprint of the specification as shown in the attached sheet (no change in content) 192-

Claims (1)

[Scope of Claims] (1) A composition containing living cells housed in a capsule made of a cross-linked protein gastric wall. (2) The composition according to claim 1, wherein the protein is selected from albumin, casein, colladan, gelatin, soy protein, gluten and immunoglobulin. (3) The composition according to claim 1, wherein the protein stomach wall further contains a water-soluble poladan compound. (4) The composition according to claim 1, wherein the protein stomach wall further contains an enzyme-degrading substance. (5) The composition according to claim 4, wherein the enzymatically degraded substance is polysaccharide, protein, or nucleic acid. (6) Cross-linked protein The composition according to claim 1, wherein the stomach wall is porous and can allow passage of cell nutrients. (7) The composition according to claim 1, wherein the cross-linked Ihitan chamber wall is porous and is capable of passing molecule i (up to 500,000 molecules or particles). The composition of claim 7, wherein the molecule is an immunoglobulin. (9) The composition of claim 8, wherein the immunoglobulin is a monoclonal antibody. 01 The composition of claim 8, wherein the molecule is a virion. The composition according to claim 7, wherein the protein stomach wall is characterized by pores with a diameter of 5X to 15 μm. (6) The cell is an animal, a plant, a microorganism, and artificial cells. (13) The composition according to claim 12, wherein the cells are hybridoma cells. The composition according to claim 12, which is a cell. α0 The composition according to claim 12, wherein the cell is a bacterial cell. αQ The composition according to claim 12, wherein the bacterial cell is 16. The composition of claim 15, wherein the composition represents a product encoded by a bacterial gene.Ofc) The composition of claim 16, wherein the non-bacterial gene is a mammalian gene. 11G The composition of claim 1, which is combined with a nutrient medium for said cells.Claim 1, wherein the nutrient medium is a test tube medium.
Composition according to item 8. (b) Claim 10 in which the nutrient medium is a biological medium.
Compositions as described in Section. QT Claim 2 in which the biological medium is animal ascites
Composition according to item 0. The composition according to claim 1, wherein the capsules have an average diameter of 250 μm or less. (a) dispersing living cells in an aqueous solution of capsule wall-forming proteins; (b) aqueous droplets of said cell-containing medium in an aqueous, miscible, cell-compatible continuous processing medium; (c) crosslinking with a crosslinking agent soluble in the processing medium and substantially insoluble in the aqueous droplets; . (c) The method according to claim 23, wherein the aqueous solution of the protein contains nutrients for the cells. (c) The protein is albumin, casein, collagen.
24. The method according to claim 23, wherein the selected ingredients are necessarily selected from Dan, gelatin, soybean tongue/ceph, gluten, and immunoglobulin. (c) The method according to claim 23, wherein the solution further contains an enzyme-degrading substance. (a) The method according to claim 26, wherein the enzymatically degraded substance is polysaccharide, protein, or nucleic acid. (c) The method according to claim 23, wherein the cells are selected from animal, plant, microorganism, and artificial cells. (c) The method according to claim 28, wherein the cells are No. ibridoma cells. (g) The method according to claim 28, wherein the cell is a bacterial cell. 31. The method of claim 30, wherein said bacterial cell represents a product encoded by a non-bacterial gene recombined with said bacterial DNA. 02. The method according to claim 31, wherein the non-bacterial gene is a mammalian gene. 03. The method according to claim 23, wherein the capsules have an average diameter of 250 μm or less. (b) The method according to claim 23, wherein the processing medium is vegetable oil or mineral oil. (→ The method according to claim 23, wherein the processing medium is selected from peanut oil, sesame oil, and cottonseed oil. The method according to claim 23, wherein the reactable at least Ninomiya substance of interest is the method according to claim 36, wherein the crosslinking agent is a non-rodanic acid. 0→ the dihalodanide. Claim 3, in which the acid has the structural formula: XOC-(CH2)□-cox (wherein,
The method described in Section 7. 24. The method of claim 23, further comprising the step of washing the crosslinked capsules with an aqueous insoluble solvent. 24. The method of claim 23, wherein the aqueous solution of θQ capsule wall-forming tannin further comprises a water-soluble poladan compound. 14]) The poladan compound is added to the solution at a concentration of 1 da to 19/
41. The method of claim 40, comprising ml. 0 埠 The poladan compound is poly(vinyl alcohol),
Cal? The method according to claim 40, wherein the poladan compound is oxymethylcellulose, poly(vinylpyrrolidone), starch or glycol. The method according to scope item 40. LL4◆ Claim 23 further comprising the step of adding a capsule wall degrading enzyme to the formed crosslinked capsule for a sufficient period of time to enlarge the pores in the wall to a predetermined average size.
The method described in section. (6) The method according to claim 44, wherein the enzyme is a proteolytic enzyme. (g) Adding a capsule wall-degrading enzyme to the formed crosslinked capsule for a sufficient period of time to enlarge the pores in the wall to a predetermined average size.
The method described in Section 7. G1) The enzyme is polysaccharide degrading enzyme, DNase,
RNase, keratinase, cellulase, amylase,
47. The method of claim 46, wherein the method is selected from coladanases. (A method for propagating cells characterized by growing 40 cells in a capsule consisting of a wall made of cross-linked tan-silicon. (6) The wall has molecules with a molecular weight of 500,000 or less and a diameter of 3,009X or less 49. A method according to claim 48, characterized in that the pores are sufficient to allow the passage of particles of. - a method according to claim 49, wherein the molecule is an immunoglobulin. 6. The method according to claim 50, wherein the cell is selected from animal, plant, microorganism, and artificial cells. 63. The method according to claim 52, wherein the cell is a virus-infected cell. ■ The method according to claim 52, wherein the cell is a virus-infected cell. Zeng: The method according to claim 52, wherein the cell is a bacterial cell. Fifth
The method described in Section 2. 56. The method of claim 55, wherein said bacterial cell represents a product encoded by a non-bacterial gene recombined with said bacterial DNA. 6'/) The method of claim 56, wherein the non-bacterial gene is a mammalian gene. 49. The method according to claim 48, wherein the cells are grown in the body of an animal. Zeng A method according to claim 58 in which the cells are grown in ascites of an animal ◇ ■ A method according to claim 58, wherein the protein contained in the membrane wall of the capsule is obtained from the animal. the method of.