JPS6038111B2 - Fixation-dependent cell culture method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention describes a method for culturing anchorage-dependent cells, i.e., cell types that are normally grown in one or more layers on a substrate and cannot be grown in suspension. Regarding.

An anchorage-dependent cell here refers to a cell that will grow in vitro only after attachment to a substrate such as dextran, DEAE cellulose or polystyrene. Anchorage-dependent cells may survive in suspension culture but do not proliferate. For example, fibroblasts, epithelial cells or smart cells are fixation-dependent cells. Advances in cell biology have shown that anchorage-dependent cells of various higher organisms produce small amounts of substances, such as interferons, that have significant potential or actual utility in disease treatment.

Such cells are also useful for research purposes. There is a risk of contamination with pre-existing microorganisms or other contamination in such cell cultures.

Also, in many cases the amount of the substance of interest produced by cell culture is extremely small. Because anchorage-dependent cells, unlike suspension cultures, cannot be cultured in large quantities, it is recognized that there is currently no practical method for growing the large numbers of cells necessary to produce the significant amounts of substances of interest produced by these cells. . Currently, anchorage-dependent cells such as normal fibroblasts and certain renal and epithelial cells must be cultured on substrates.

Plastic sheets used as substrates are commercially available. Certain anchorage-dependent cells proliferate until this sheet is covered with a monolayer, and then stop proliferating. Other cells continue to grow, adding 1 or 2 more cells on top of the first layer.
Form the above layers. The present invention provides a method for culturing fixation-dependent cells in suspension, which allows for the growth of larger amounts of cells per unit volume than in monolayer culture.

According to the present invention, a culture serving as a source of fixation-dependent cells is filled and encapsulated in a plurality of microcapsules made of semipermeable membranes. The membrane synthesis is adjusted so that the membrane has a selected upper permeability limit. That is, if the membrane is e.g.
The synthesis of the membrane is adjusted to define micropores large enough to pass molecules having molecular weights up to 1.times.x1 and Daltres, but to block the passage of higher molecular weight substances. The capsule membrane defines a microenvironment that confines the cells with high molecular weight serum components. However, this microenvironment is one that allows the cells access to low molecular weight cellular nutrients such as amino acids and allows the efflux of low molecular weight cellular metabolites out of the environment. The microcapsules containing cells are then dispersed in a conventional culture medium.

Certain high molecular weight water-loving substances contained within the inner surface of the capsule membrane and/or within the microcapsules serve as a substrate to which cells attach. Because the ratio of the surface area of the capsule to the volume of medium outside the capsule is extremely high, each cell has sufficient access to the necessary nutrients, and the area on which the cells can grow is increased compared to conventional monolayer culture. in general,
The average diameter of the microcapsules can vary between a few micrometers to one rib or more. The preferred average size is 10 in diameter.
It is 0 to 500 Buddhist pedestals. When a fixed matrix such as collagen is included within the capsule, fibroblasts also form inside the capsule membrane and assume normal fibroblast morphology. It is an object of the present invention to provide an improved method for culturing anchorage-dependent cells.

Another objective is to improve the yield of fixation-dependent or contact-inhibited cells grown in vitro. Another objective is to provide a large surface area suitable as a substrate for anchorage-dependent cells throughout the volume of the cell growth medium. Another object is to provide a method for growing cells capable of producing interferon. These and other objects and features of the invention will become apparent from the following description and the drawings, the only illustration of which is a photograph (200x magnification). This photograph shows anchorage-dependent cells grown in microcapsules according to the present invention and establishes the traditional fibroblast morphology. The broad concept of the invention is to provide multiple semipermeable membranes around individual cells or groups of cells to act as a large surface area matrix to which the cells can adhere, and to encapsulate high molecular weight substances within the microcapsules. It is to confine it and make it act as a fixed substrate.

Cells that grow in suspension can also be encapsulated as taught herein. The microcapsules, together with at least the high molecular weight components of the medium, serve as a microenvironment for the cells and separate the cells from the extracapsular aqueous medium. Microorganisms and other relatively large contaminants cannot pass through this membrane. Anchorage-dependent cells of southern mammalian origin, such as stroblasts, epithelial cells, or cells, require the presence of serum components to continue growing.

Some of this component has a molecular weight that exceeds the permeability limit of the membrane. Such components may be included with the encapsulated cells, but need not be present in the medium outside the capsule. Additionally, the natural protein collagen, which is the main component of connective tissue, has been successfully used as a high molecular weight substance that acts as a fixation matrix within microcapsules. Other compatible high molecular weight hydrophilic proteins such as polylysine may also be used. If the protein has free amino/groups, it can be made water-shedtable by reacting with a water-soluble gum during membrane formation, as described below. The use of such materials is believed to create a matrix within the capsule. By including such substances, it is possible to increase the concentration of cells within the capsule when cells grow inside the capsule instead of or in addition to growth on the inner periphery of the capsule membrane. . The encapsulated source culture is then suspended in a suitable growth medium of the type used for growing conventional cultures.

Serum proteins not taken up by the cells may be removed from the extracapsular medium. However, the pH, temperature, ion concentration, etc. should be the same as in conventional media. oxygen and C
02, migration can be promoted in the same way as in conventional culture, as these dissolved gases freely permeate the membrane. Incubation (flooding) of the encapsulated cell culture results in mitosis of the cells. The growth of linear longitudinal blastocytes takes on the traditional fibroblastic morphology, forming cell rows on a fixed matrix contained within a membrane or capsule. Fresh growth medium is provided by changing the medium outside the capsule, either continuously or intermittently as necessary. When the purpose of the culture is to produce a cellular metabolite of interest, the metabolite is obtained either from the capsule contents or from the extracapsular material, depending on the molecular weight or upper permeability of the membrane. In a preferred embodiment of the method, the membrane is of a type that can be selectively disrupted without harming the cells. This membrane allows cells to be released from the capsule when desired. One of the reasons for releasing cells after the growth phase is to stimulate the production of substances of interest by the cells.

An example is the production of interferon from human fibroblast, leukocyte, or lymphoblast cells. Treatment of these cells with certain viruses or high molecular weight nucleic acids induces interferon excretion. In this situation, if the upper permeability of the membrane is below the high molecular weight of the inducer, cells can be subjected to interferon-transfer treatment before encapsulation, or the capsule membrane can be selectively transfected after culturing the cells. They must be broken to allow these high molecular weight substances to come into contact with the cells. The method of the invention relies on the ability to form a semipermeable membrane around the cell without simultaneously adversely affecting ongoing growth.

One method of suitable encapsulation is detailed below. Encapsulation of Cells The tissue or cells to be encapsulated are suspended in a preferably aqueous matrix for growth of the type of cells involved.

Media suitable for this purpose are commercially available. The average diameter of the material to be encapsulated can vary widely between a few r and about one rib. However, best results are obtained with capsule sizes ranging from 100 to 500 Buddhas. Individual anchorage-dependent cells such as broodoblasts, epithelial cells, and epithelial cells of human or animal origin may be encapsulated if desired. Cells such as leukocytes, lymphoblasts, total beta cells, alpha cells, delta cells, mixtures of these in various proportions, or other tissue units may also be encapsulated. The ongoing growth potential of such organisms depends, inter alia, on the availability of nutrients, oxygen transfer, the absence of toxic substances in the medium, and the pH of the medium.

Hitherto, it has not been possible to maintain such living organisms in a physiologically compatible environment while simultaneously encapsulating them. The problem is that the conditions required for membrane formation are lethal or harmful to the tissue, and that U.S. Patent Application No. 953,4
Prior to the inventions of Nos. 13 and 24, and 60, there was no pus formation method that would keep tissues alive in a healthy state. However, it is physiologically compatible with living tissue,
Certain hydrophilic substances that can form water-tight, shape-retaining aggregates are used to create "temporary capsules" or protective barrier layers around individual cells or groups of cells. It has been found that this temporary capsule can be formed and that this temporary capsule can be manipulated to provide a more permanent semi-permeable wall around the cell without damaging the cell.

Such materials typically include 1. is added to the tissue culture medium at concentrations on the order of % by weight or less. The medium further contains a source culture, serum components (if necessary), and optionally collagen or other high molecular weight water-soluble substances to act as immobilization substrates. The concentration of the material used as a substrate should be in the range of about 10 to 9/1, but preferably 100 to 500 mg/. This solution is then formed into droplets containing the tissue and its medium, and immediately at least the surface layer is insolubilized and gelled in water. The shape-retaining temporary capsule is then formed with a more permanent membrane that can subsequently be selectively ruptured when release of the intact tissue is desired. If the material used to form the temporary capsule is permeable, the interior of the capsule may be reliquefied after the permanent membrane is formed. This reliquefaction is carried out by creating conditions in which the substance becomes soluble again in the deposit. The material used to form the temporary capsule can be any non-toxic, water-soluble material that can be transformed into a shape-retaining object by a change in ionic environment or concentration.

The material should further contain a plurality of ionizable anionic residues, such as carboxyl groups, which can react with polymers having a plurality of ionic groups by forming salts. As discussed below, the use of this type of substance
The upper permeability limit (generally 100,000 to 150,000
Permanent membranes can be provided with a diameter of less than 0 Daltons). Preferred materials for forming general capsules in the present invention are acidic, water-soluble, natural or synthetic polysaccharide gums.

Such materials are commercially available. These substances are typically extracted from plant matter and then used as additives in various foods. Sodium alginate is a preferred water-soluble gum in the present invention. 150,
Alginates with molecular weights in the range of 1,000 Daltons can also be used, but their molecular size and viscosity usually make it impossible to penetrate finely formed capsule membranes.

Lower molecular weight alginates, e.g. 50,000~
80,000 Daltons are preferred because they are easily removed from the capsule interior by diffusion through membranes with sufficient porosity. Other gums that can be used include the acidic fraction of guar gum, carrageenan, pectin, tragacanth gum, or xanthan gum. These substances contain glycosidic sugars. These free acid groups now exist in the alkali metal ion form, such as the sodium form. When alkali metal ions are replaced with multivalent ions such as calcium or strontium, water-soluble polysaccharide molecules are "crosslinked" to form a gel that retains its water-insoluble form. This gel can be resolubilized upon removal of ions by ion exchange or by using sequestering agents. Although essentially any multivalent ion capable of forming salts with acid gums can be used, it is preferred to use physiologically compatible ions, such as calcium. The use of such ions helps preserve tissue in a viable state. Other multivalent ions can also be used. Magnesium ions are not effective in gelling sodium alginate. Typical solution compositions include cell cultures in soil (with or without immobilized substrate);
and a 1-2% gum solution in physiological saline.

When using sodium alginate, 1.0-1.5%
Solutions are convenient for use. Collagen or polypeptides, either natural or synthetic, can be included in the cell culture and ultimately entrapped within the capsule that forms. When a polymer having multiple anionic groups, such as polylysine, is used, the cationic groups react with the anionic sites of the water-soluble rubber, resulting in a substantially water-insoluble material that is entangled with the rubber. form a matrix of Preferred concentrations of such materials are on the order of 100-500 rg per suspension (including gummy solutions). In the next step of the encapsulation process, the gummy solution containing the tissue is formed into droplets of the desired size and immediately gelled to yield shape-retaining spherical or elongated objects.

Droplet formation is performed as follows. A tube containing an aqueous solution of polyvalent cations, for example a 1.5% CaC12 solution, is fitted with a stopper that supports the droplet forming device. This device consists of a jacket with an upper intake nozzle and an elongated hollow friction element fixed on the stop. A 10 cc syringe with a step pump is attached to the jacket, for example, by a Teflon-coated needle with an internal diameter of 0.254 skin (0.01 inch) extending longitudinally through the jacket. The interior of the jacket 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, the syringe is filled with a solution containing the substance to be encapsulated and the tip pump is activated to incrementally expel droplets of solution from the tip of the needle. Each droplet is "cut" by the airflow and approximately 2. & fell off the pillow C
Enter the aC12 solution. In this solution, the droplets immediately gel due to the absorption of calcium ions. The tip of the needle and C
The distance between the aC12 solution and the surface is in this case large enough to give the sodium alginate/cell suspension the physically most favorable shape, ie spherical (maximum volume with minimum surface area). Air within the tube flows through a closure within the stopper. This causes the gel to "bridge" and form a temporary capsule that retains a highly viscous shape containing the suspended tissue and medium. The capsules collect as separate layers in the solution and are separated by suction. The next step in the method is to provide a semi-permeable membrane around the surface of the temporary capsule by means of a "bridging" surface layer.

This step is carried out by treating the gelled temporary capsule with an aqueous solution of a polymer containing enteric ionic groups that react with anionic functional groups within the gel molecule. Polymers containing acid reactive groups such as free imine or amine/groups are preferred. In this case, the polysaccharide gums are cross-linked by interactions between carboxyl groups and amine or imine groups (formation of salt bonds). selecting the molecular weight of the crosslinked polymer used;
And the permeability can be controlled by adjusting the concentration of the polymer solution, treatment time and temperature. A polymer solution with a lower molecular weight will penetrate into the temporary capsule more than a polymer with a higher molecular weight in a given time. The degree of penetration of the crosslinking agent is correlated with the degree of penetration obtained. Generally, the higher the molecular weight, the lower the permeability and the higher the porosity.
Polymers in the molecular weight range of 3,000 to 100,000 daltons can be widely used depending on the time of reaction, concentration of the polymer solution, and desired permeability. Approximately 35,0
One combination of effective reaction conditions using polylysine with an average molecular weight of 0.00 Daltons is to react for 2 minutes with physiological saline containing 0.0167% polylysine. This reaction produces a membrane with an upper permeability of about 100,000 Daltons. The optimal reaction conditions used to adjust permeability within a given system can be easily determined empirically from the above guidelines. Using this method,
It is possible to set the upper permeability of the membrane to a selected level, generally below about 150,000 Daltons. Examples of suitable cross-linked polymers include proteins and polypeptides, either natural or synthetic, having free amino or imino groups, polyethyleneamine, polyethyleneimine, and polyvinylamine.

Both the D and L forms of polylysine are useful. Proteins such as polyarginine, polycitrulline, or polyornithine may also be used. Polymers in the high positive charge density range, such as polyvinylamine, rapidly adhere to the anionic groups of gel molecules to form a stable film that is resistant to premature tearing. Treatment with dilute solutions of gummy or zwitterionic buffers fixes free amino groups on the capsule surface, which may give the capsules a tendency to agglomerate.

At this point in encapsulation, a capsule containing a semipermeable membrane surrounding the gummy gelling solution, a culture medium compatible with the cell type, the cells, and optionally an inner matrix of collagen or other fixed matrix can be recovered. .

Since mass transfer should take place within the capsule and through the pus, it is preferable to reliquefy the gel to a water-soluble form. This operation is carried out by re-establishing conditions in which the gum becomes liquid, eg, conditions that remove calcium or other polyvalent cations from the internal gel. The medium within the capsule can be resolubilized by simply soaking the capsule in phosphate buffered saline containing alkali metal ions and hydrogen ions. When the capsule is immersed in a solution, the monovalent ions undergo ion exchange with calcium or other multivalent ions. A sodium citrate solution can be used for the same purpose, and this solution acts to mask divalent ions. The encapsulated cell culture as described above is suspended in a growth tower specially prepared to meet all the needs of the particular cell type involved and allowed to undergo normal in vitro metabolic and mitotic progression. let it continue.

If the medium requires an environment of high molecular weight components such as serum components, these components are removed from the medium outside the capsule.
Typically, the components normally ingested by cells are of relatively low molecular weight and readily diffuse through the capsule membrane into the cell's microenvironment where the components permeate the cell membrane. Cellular metabolites excreted into the medium within the capsule will simultaneously diffuse through the membrane and aggregate within the medium outside the capsule, if these products have a molecular weight below the upper permeability limit of the capsule membrane. do. Encapsulated cells are grown, for example, under the same temperature, vessel, and ionic environment conditions as conventional culture media. Cellular metabolites are obtained from the medium outside the capsule or from inside the capsule by conventional methods. However, the culture method described herein has the following advantages. 1 Cells in culture are protected from contamination by factors whose size exceeds the upper permeability limit of the membrane.

This means that the need for sterilization, which is normally inherent in culturing methods, is greatly alleviated since other microorganisms cannot access the encapsulated cells. 2 The capsule effectively immobilizes the cells in an environment that:

That is, this environment is one that confines the high molecular weight substances involved, yet allows for the easy removal and introduction of low molecular weight cell nutrients and products. This environment allows for intermittent or continuous withdrawal and replenishment of the nutrient tower, if desired, without damaging the cells. 3. Substances of interest produced by cells are more easily recovered.

Cellular products with a molecular weight small enough to penetrate the capsule membrane collect in the extracapsular reservoir in a mixture with nutrients. However, high molecular weight serum components etc. are not released into the medium outside the capsule, thus simplifying the recovery of the cell products of interest. Cellular products with molecular weights above the permeability limit of the membrane collect within the capsule. Such products are recovered in a relatively concentrated state, for example, by isolating the capsules and subsequently selectively rupturing the membranes using the methods described below. 4. The inside of the capsule provides a sufficiently suitable environment for cell division.

It is observed that the suspension culture continues to undergo mitosis within the capsule. Anchorage-dependent cells, which grow as two-dimensional monolayers in normal culture, proliferate to form columns within the capsule. Such cells use the inner surface as a matrix and attach to the above-mentioned high molecular weight materials provided within the capsule. Therefore, the cell density is significantly higher than in conventional culture. The continued growth potential of such cell colonies is supported by the fact that the ratio of surface area to volume of the capsule is extremely high, so that all the cells have access to the necessary nutrients, oxygen, etc. In some cases, it is advantageous to selectively rupture the capsule membrane to release the cells without causing damage.

One notable example is interferon (INF).
This is the case for the production of Cells capable of producing WF must be treated with certain viruses or nucleic acids in preparation for the mF production step. Additionally, several Kawa F-guided methods include adding reagents to the culture to inhibit protein synthesis. Therefore,
The growth stage of the culture process must be carried out under very different conditions than the INF concession stage. ...If the substance used for F induction has a molecular weight that exceeds the upper permeability limit of the capsule membrane (as in the case of virus induction), the induction step cannot be carried out in the encapsulated cell culture. Therefore, m
Once the F-producing cells have grown within the capsule, they should be released and subjected to the induction step by rupturing the membrane. Disruption of Membranes Cells entrapped within membranes of the type described above can be released in a manner that includes commercially available agents that have the property of not having significant adverse effects on the encapsulated cells.

First, the capsules are separated from the suspension medium, thoroughly washed to remove contaminants present on the outside of the microcapsules, and then washed with monoatomic polyvalent cations such as calcium ions and polysulfone. It is dispersed in a mixed solution with a polymer having multiple anionic residues, such as a salt of acid or polyphosphoric acid. Heparin, a natural sulfonated polysaccharide, is preferred for this step. The anionic charge density of the polymer used should be equal to, or preferably greater than, the charge density of the acidic rubber originally used to form the membrane. The molecular weight of the polymer should be at least comparable to, and preferably greater than, the molecular weight of the polymer with multiple enteric ionic groups used for membrane formation. Inside the capsule suspension in the mixed liquid, calcium ions compete with the ionic polymer chains used for pus formation for anionic sites on the water port.
At the same time, the anionic gums of the membrane compete for cationic sites on the polymer chains with heparin or other polymers with multiple anionic residues dissolved in solution. This results in, for example, water-dispersible or preferably water-soluble complexes of polylysine and heparin, and the association of monovalent cations with molecules of the gel. This step allows the membrane to dissolve upon subsequent exposure to a sequestering agent.

This sequestering agent completes the destruction process by removing monovalent ions from the gel. Any capsule membrane fragments remaining in the culture medium can be easily separated from the cells. A generally preferred solution for use in the first stage of the selective destruction process contains 1.1% (w/v) calcium chloride and between 500 and 1,50 units of heparin per Z of solution.

The contents of the microcapsules are added to this solution in such a way that they represent between approximately 20% and 30% of the total volume of the suspension. Strontium salts or other multivalent cations (with the exception of Mg deca ions) are preferred as they are compatible with the above-mentioned types of polysulfonates or polyphosphates, and therefore pose less risk of cell damage. Generally, the concentrations of monovalent ions and anionic polymer used in this step can vary over a wide range.

Optimal concentrations can be easily determined empirically and depending on the exposure time and the particular polymer used to form the film. The generally preferred sequestering agent for selective destruction is sodium citrate, although other alkali metal salts of citric acid and EDTA alkali metal salts may also be used.

When using sodium citrate, the optimal concentration is 50-6
It is on the 0mM level. It is preferred that citrate or other sequestering agents be dissolved in isotonic saline to reduce cell damage. The present invention will be explained in detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 Human fibroblasts were obtained by treating a conventional monolayer culture with tryptophan and EDTA at 370° C. for 5 minutes in a known manner to obtain 40% (V/V) purified fetal bovine serum, 0.8% sodium alginate (Sigma) and 2
The sample was suspended in a complete growth matrix (CMLRI 969, Connaught Laboratories) supplemented with 00 g/' of purified calfskin collagen.

The density of the cell suspension is approximately 1.5 x 107 cells/cell.
A 1.5% solution of calcium chloride is then used to form a vial using a droplet forming device as described above.

Droplets on the order of 50-500 mm in diameter immediately gel when they leave the needle tip and enter the calcium solution. 5 minutes later,
Aspirate and remove the top liquid. Then 0.6% NaC1 diluted with 1% CaC122 base (isotonic solution, pH=8.2
Transfer the gelled capsules to a Z beaker of Solution 15 containing 1 part of 2% 2-(cyclohexylamino)ethanesulfonic acid buffer solution in ). The capsules are then transferred into a solution 32' containing 0.005% (w/v) polylysine (average molecular weight 43,000 daltons) in physiological saline.

After 3 minutes, the polylysine solution is passed through a fugata oven.

Subsequently, the resulting capsules with a permanent semi-permeable membrane are washed twice with 1% CaC and twice with physiological saline and mixed with a 0.03% solution of sodium alginate. The gel inside the capsule inhibits aggregation and is thought to contain all cytoblasts.
Reliquefy by soaking for 5 minutes in pH 7.4). All the above steps are carried out at 22-370 °C. Under the microscope, it is observed that these capsules contain a very thin membrane surrounding the cells.

Molecules with molecular weights up to about 100,000 are permeable through this membrane. The resulting capsules are suspended in CMLR-1969 supplemented with 10% fetal bovine serum.

Microscopic observation of the capsules after 4-5 days of culture at 370°C revealed that the capsules contained postmitotic holoblasts and exhibited traditional holoblast morphology within the microcapsules. do.

By settling a portion of the microcapsule suspension 10 containing about 500-1000 capsules/secretion, the capsule membrane can be ruptured without damaging the cells.

After removing the medium with suction, the capsules are washed twice with saline.
The washed capsules are then mixed with 3.0' aliquots containing 100 units/c of heparin and 1.1% (w/v) CaC12. The suspension is stirred at 37°C for 3 minutes, after which the capsules are allowed to settle, the supernatant liquid is filtered off by suction, and the capsules are washed twice with 0.18M NaCl 13.0. After passing the second washing solution through a suction oven, 11M sodium citrate and 0.19M Na
A mixed solution containing an equal volume of CI (pH=7.4) 2.0% is mixed with the capsules.

The mixture was manually swirled to break up the membrane;
Wash cells twice with medium. Linear holoblasts are Vilsek (V
INF-B superinduction treatment according to the INF-B method (ilcek).

P.L. Biochemicals, Milwaukee, Wisconsin, in a 5% C02 atmosphere (95% air).
Polyl-
PolyCIOO Buddhag / Secretion and Cycloheximide [Calbioc, La Jolla, California]
The above cell suspension is cultured at 37°C for 1 hour in the presence of 50 g/g/' of a protein synthesis inhibitor of hem).

After 1 hour, washed in medium containing 50 rg/' of cycloheximide (CMLR-1969) and then washed with 5% C0
Suspend in the same solution for 3 hours at 370°C under 2 atmosphere.

When this incubation was completed, the washing step was repeated, and 50 g/g of cycloheximide and 51 g/g of actinomycin D (a known RNA synthesis inhibitor from Calbiochem) were added.
and cultured for 2 hours at 37°C in a 5% CO2 atmosphere. Cells were then washed twice in medium and grown for 18 min at 37o0 in serum-free medium.
Suspend for ~2 hours, during which time the trichoblasts become INF.
secrete 18. INF-8 has a molecular weight on the order of 21,000 daltons and can be obtained from extracapsular beaches. Sedimentation coefficient Polyl-PoclyC (Poly
In an experiment using INF-8 (in which double helical RNA was formed by annealing of INF-1 and PolyC to form a double helical RNA), 2,50 INF-8 peaks per cell in the culture.
occurred.

The same yield was obtained in a second experiment using the same PoM-Pol length (double helix when obtained).

Other embodiments are within the scope of the claims.

[Brief explanation of the drawing]

FIG. 1 is an enlarged photograph of cells grown in microcapsules. Figure 1

Claims (1)

[Scope of Claims] 1. A method for culturing fixation-dependent cells, the method comprising A. suspending said cells in a medium containing an immobilized substrate material and a polymeric component (A), the molecular weight of which exceeds a selected level. Make it muddy, B
said component (A) and said immobilized matrix material within a plurality of semipermeable membranes having an upper limit of semipermeability sufficient to prevent the permeation of said component (A) and said immobilized matrix material, and to allow molecules having a molecular weight below said selected level to permeate through the membrane. Cells are encapsulated with said medium containing said immobilized matrix material, component (B) consisting of amino acids, salts and vitamins that can be assimilated by the C cells, provided that the molecular weight of said component (B) is below said selected level. Step B to the medium containing
A culture method comprising the steps of suspending the product of D and subjecting said cells to mitosis within said membrane. 2 Claim 1 in which the immobilized substrate is a protein
The method described in section. 3. Claim 1, wherein the fixed matrix is collagen.
The method described in section. 4. The method of claim 1, wherein the fixed substrate is calfskin collagen and is included in the suspension at a concentration between about 10 μg/ml and 1.0 mg/ml. 5. A method for culturing fixation-dependent cells, wherein the method comprises A. suspending said cells in a medium containing an immobilized substrate material and a polymeric component (A), the molecular weight of which exceeds a selected level;
said component (A) and said immobilized matrix material within a plurality of semipermeable membranes having an upper limit of semipermeability sufficient to prevent the permeation of said component (A) and said immobilized matrix material, and to allow molecules having a molecular weight below said selected level to permeate through the membrane. Cells are encapsulated with said medium containing said immobilized matrix material, and C cells are encapsulated in a medium containing component (B) consisting of amino acids, salts and vitamins assimilated by the cells, provided that the amount of said components is below said selected level. suspending the product of step B; D subjecting said cells to mitosis within said membrane;
and E. A culture method consisting of various steps in which cells are selectively covered with a membrane and released. 6. The method according to claim 1, wherein the cells consist of fibroblasts. 7. The method according to claim 1, wherein component (A) consists of a serum component. 8. The method according to claim 1, wherein the cells consist of human fibroblasts capable of secreting interferon. 9. A method for culturing fixation-dependent cells, wherein the method comprises A. suspending said cells in a medium comprising an immobilized substrate material and a polymeric component (A), the molecular weight of which exceeds a selected level;
said component (A) and said immobilized matrix material within a plurality of semipermeable membranes having an upper limit of semipermeability sufficient to prevent the permeation of said component (A) and said immobilized matrix material, and to allow molecules having a molecular weight below said selected level to permeate through the membrane. Cells are encapsulated with said medium containing said immobilized matrix material, component (B) consisting of amino acids, salts and vitamins that can be assimilated by the C cells, provided that the molecular weight of said component (B) is below said selected level. Step B to the medium containing
suspending the products of D, allowing the cells to undergo mitosis within the membrane, E selectively rupturing the membrane to release the cells, and F
Cells are subjected to interferon induction treatment, G. Step F
A culture method consisting of various steps of culturing cells produced in a medium in a medium, and obtaining interferon from the medium in Step G. 10. The method of claim 1, wherein during the encapsulation step a spheroidal membrane having an average diameter in the range 100-500μ is formed. 11. The method according to claim 1, wherein the immobilized substrate consists of a protein having a plurality of free cation groups. 12. The method of claim 1, wherein the selected level is less than or equal to about 2.0 x 10_5 Daltons. 13. The method of claim 1, wherein the selected level is less than or equal to about 1 x 10_5 Daltons.