CA1172586A - Method of culturing anchorage dependent cells - Google Patents

Abstract

METHOD OF CULTURING ANCHORAGE DEPENDENT CELLS

Abstract of the Disclosure Disclosed is a method of growing anchorage-dependent cells: cells of the type which normally undergo mitosis only when anchored on a substrate, e.g., fibroblasts or epithelial cells. The method comprises the steps of encapsulating a seed culture of the cells within a semipermeable membrane and suspending the capsules in a growth medium. The interior sur-faces of the capsule membrane and/or collagen enclosed within the capsules serve as a substrate for the cells. The ratio of the available substrate surface area to the volume of the culture may be large, thereby allowing the cells to be grown substantially throughout the volume of the culture medium.

Description

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_, ( , . _ ~L3 72S86 1 Summary of the Invention This invention provides a method of culturing anchorage dependent cell~ in su~pension and thereby enables greater numbers of cell~ to be grown per unit volume of culture medium as com-pared with monolayer cultures.

In accordance with the invention, a seed culture of anchorage dependent cells iB encapsulated within a plural$ty of microcapsule6 comprising ~emipermeable membranes. Synthesis of the membranes is controlled 80 that they have a selected upper limit of permeability, that i~, the membranes define micropores of dimensions sufficient to allow passage of molecules having a molecular weight up to, for ex~mple, 2x105 daltons, but substan-tially preclude passage of materials of higher molscular weight.
The capsule membrane thus defines a microenvironment within which the cell together with high molecular weight ~erum components are confined, but which allow the cells free acce~s to lower molecular weight cell nutrients such a~ amino acid~, and allow low molecular weight cell metabolites to exit the environment.

The microcApsules containing cell~ are then dispersed in a conventional culture medium. The interior ~urfaces of the cap~ule membrane and/or certain high molecular weight water-disper~ible materials which are includ~d ln the m~crocap~ule ~ct as a ~ubstrate to which the cells attach. Bec~u~e the ratio of the aurface area of the c~psulea to the volume of the extracap-sular medium may be quite large, individu~l cells are afforded adequ~te acc~s to required nutrients, and the ~rea on which the cell~ can grow iB increased a~ compared with conYentional mono-layer cultures. Generally, the ~verage diameter of the microcap-:1~7~586 1 sules may be varied between a few microns to a millimeteror more. A preferred average size is on the order of 100-500 micrometers in diameter.
If an anchorage substrate such as collagen or the like is included within the capsules, then fibroblasts also grow inwardly of the capsule membranes and will display normal fibroblastic morphology.
It is an object of the invention to provide an improved method of culturing anchorage dependent cells.
Another object is to improve the yield of anchorage dependent or contact inhibited cells grown in vitro. Another object is to provide throughout the volume of a cell growth medium a large surface area suitable as a substrate for anchorage dependent cells. Another object is to provide a method of growing cells which can produce interfereon. These and other objects and features of the invention will be apparent from the description which follows and from the drawing wherein the sole figure is a photomicrograph (200X) showing anchorage dependent cells grown within a microcapsule in accordance with the invention and illustrating classical fibroblastic morphology.

1~72586 1 Descrip-tion The broad concept of the invention is to provide a multiplicity of semipermeable membranes about individual cells or groups of cells to act as a high surface area substrate to which the cells can anchor and/or to confine a high molecular weight material within microcapsules to serve as an anchorage substrate. Cells which grow in suspension may also be encapsulated as taught herein and in the above-referenced copending applications. The microcapsules serve as a microenvironment for the cells together with at least high molecular weight components of its culture medium, and separate the cells from an extracapsular aqueous medium. Bacteria and other relatively large contaminants cannot penetrate the membranes.
Anchorage dependent cells of mammalian origin, such as fibroblast, epithelial, or kidney cells require for ongoing viability the presence of serl~ components, a portion of which may have a molecular weight in excess of the upper per-meability limit of the membranes. Such components may be included with the encapsulated cells and need not be presentin the extracapsular medium. -It is required also to include within themicrocapsules a high molecular weight material to serve as an anchoring substrate. Collagen, a natural protein which is a major constituent of connective tissues, has been used with success for the purpose. Other compatible high molecular weight, water dispersible proteins may also be used, e.g., polylysine. If the proteins have free amino groups, they may be rendered water-insoluble by reaction with a water-soluble gum during membrane formation as disclosed hereinafter. The ~72586 1 use of such materials is believed to result in the creation of a matrix within the intracapsular volume. The inclusion of such a material can improve the cell density within the cap-sules as the cells grow within the intracapsular volume instead of or in addition to the growth about the interior of the capsule membrane.
The encapsulated seed culture is then suspended in a suitable growth medium of the type employed for growth of con-ventional cultures. Serum proteins which are not injested by the cells may be omitted from the extracapsular medium.
However, pH, temperature, ionic concentration, and the like should be the same as in conventional media. Also, oxygen and C02 transfer may be promoted by the same means as in conventional cultures, as these dissolved gasses freely traverse the membrane.
Incubation of the encapsulated cell culture results in cell mitosis. Fibroblast cell growths display classical fibroblastic morphology and form arrays of cells on the interior of the membrane or on the anchoring substrate contained within the capsules. Fresh growth medium may be supplied as required either on a continuous or intermittent basis by change of the extracapsular medium. If the purpose of the culture is to produce a cell metabolite of interest, the metabolite may be harvested either from the intracapsular volume or the extracapsular medium, depending on its molecular weight and the upper permeability limit of the membranes (see copending Canadian application Serial No. 398,218. In a preferred embodiment of the process, the membranes are of a type which may be selectively disrupted without damage to the cells.
This allows the cells to be released from the capsules as ,. . .

1 desired (see copending Canadian application Serial No. 398,210).
One reason for releasing the cells after their growth period is to stimulate the production of a substance of interest by the cells. An example is the production of interferon from human fibroblasts, leukocytes, ox lymphoblastoid cells which are induced to excrete interferon by treatment with certain viruses or high molecular weight nuclei acids. In such a situation, if the upper permeability limit of the membranes is less than the molecular weight of the inducing factor, the cells must be sub~ected to interferon induction prior to en-capsulation, or the capsule membranes, after culture of the cells, must be selectively disrupted to allow such high molecular weight materials to come into contact with the cell.
The process of the invention depends on one's ability to form semipermeable membranes about cells without simultaneously adversely affecting their ongoing viability.

One suitable encapsulation process is set forth in detail below.
Cell Encapsulation The tissue or cells to be encapsulated are suspended in an aqueous medium preferably suitable for growth of the cell type involved. Media suitable for this purpose are available commercially. The average diameter of the material to be encapsulated can vary widely between a few micrometers to about a millimeter. However, best results are achieved with capsules of a size in the range of 100-500 micrometers. Indi-vidual anchorage dependent cells such as fibroblasts from human or other animal tissues, kidney cells, and epithelial cells may be encapsulated as desired. Also another cells such ,~

~172586 1 as leukocytes, lymphoblastoids, pancreatic beta cells, alpha cells, delta cells, various ratios thereof, or other tissue units may be encapsulated.
The ongoing viability of such living matter is dependent, inter alia, on the availability of required nutrients, oxygen transfer, absence of toxic substances in the medium, and the pH of the medium. Heretofore, it has not been possible to maintain such living matter in a physiologically compatible environment while simultaneously encapsulating.
The problem has been that the conditions required for membrane formation have been lethal or harmful to the tissue, and prior to the invention of Canadian application Serial No. 348,524, now Patent No. 1,145,258, no method of membrane formation which allowed tissue to survive in a healthy state had been forthcoming.
However, it has been discovered that certain water-soluble substances which are physiologically compatible with living tissue and can be rendered water-insoluble to form a shape-retaining, coherent mass, can be used to form a "temporary capsule" or protective barrier layer about individual cells or groups of cells and that this temporary capsule can be treated to deposit a more permanent semipermeable membrane about the cells without damage to the cells. Such a substance is added, typically at a concentration on the order of less than 1.0 weight percent, to the tissue culture medium which also con-tains cells of the seed culture, serum components (if required), and collagen or another high molecular weight, water-dispen-sible material which acts as an anchoring substrate. The g _ ,~

- ( 1 concentration of the material employed a8 ~ substrate ~hould be within the range of about 10 ug/ml to about 1 mg/ml but is pre-ferably on the order of 100-500 ug/ml.

The solution i~ then formed into droplets containing tissue together with its medium and iB immediately rendered water-insoluble and gelled, at least in a surface layer.
Thereafter, the shape-retaining temporary cap~ule~ are provided with a more permanent membrane which may itself ~ubsequently be selectively disrupted if it is desired to release the tisaue without damage. Where the material used to form the temporary capsules permits, the capsule interior may be reliquified af~er formation of the permanent membrane. This i8 done by re-e~tablishing the conditions in the medium at which the material is soluble.

The material used to form the temporary capsules may be any non-toxic, water-soluble material which, by a change in ionic environment or concentration, can be converted to a shape-retaining mass. The material ehould also contains plural, easily ionized anionic moieties, e.g., carboxyl groups, which can react by salt formation with polymers containing plural cationic groups. As will be e~plained below, use of thi6 type of material enables one to deposit a permanent membrane of a ~elected upper limit of permeability (generally no greater than 100,000 to 150,000 daltons) without difficulty in ~urface layer~ of the tem-porary cap~ule.

The presently preferred materials for forming the tem-porary capsule are acidic, w~ter-~oluble, natural or synthetic _ ~7~5136 1 polysaccharide gums. Such materials are commercially available. They are typically extracted from vegetable matter and are often used as additives to various foods. Sodium algi-nate is the presently preferred water-soluble gum. Alginate in the molecular weight range of 150,000+ daltons may be used, but because of its molecular dimensions and viscosity will usually be unable to permeate the finally formed capsule membranes. Lower molecular weight alginate, e.g., 50,000-80,000 daltons, is more easily removed from the intra~
capsular volume by diffusion through a membrane of sufficient porosity and is therefore preferred. Other useable gums include acidic fractions of guar gum, carageenan, pectin, tragacanth gum, or xanthan gum.
These materials comprise glycoside-linked saccharide chains. Their free acid groups are often present in the alkali metal ion form, e.g., sodium form. If a multivalent ion such as calcium or strontium is exchanged for the alkali metal ion, the water-soluble polysaccharide molecules are "cross~linked"
to form a water-insoluble, shape-retaining gel which can be resolubilized on removal of the ions by ion exchange or via a sequestering agent. While essentially any multivalent ion which can form a salt with the acidic gum is operable, it is preferred that physiologically compatible ions, e.g., calcium, be employed. This tends to preserve the tissue in the living state. Other multivalent cations can be used. Magnesium ions are ineffective in gelling sodium alginate.
A typical solution composition comprises equal volumes of a cell culture in its medium ~with an anchoring sub-strate) and a one or two percent solution of gum in physiolo-~7i~586 1 gical saline. When employing ~odium algina~e, a 1.0 to 1.5 per-cent solution has been used with BUCce~B. Collagen or another high molecular weight water-dispersible protein or polypeptide, either natural or synthetic, may be included in the cell culture, and will be confined within the intracapsular volume of the finally formed capsules. If a polymer having plural cationic groups i8 employed, e.g., polylysine, the cationic groups react with anionic sites in the water-soluble gum to form a substan-tially water-insoluble matrix intertwined with the gum.
Preferred concentrations for such materials are on the order of 100-500 ug per ml of suspension (including gum solution).

In the next ~tep of the encapsulation process, the gum aolution containing the ti~sue i~ formed into droplet~ of a de6ired size, and the droplets are immediately gelled to form shape-retaining spherical or spheroidal masses. The drop forma-tions may be conducted as follows.

A tube containing an aqueous solution of multivalent cations, e.g., 1.5% CaC12 solution, iB fitted with a stopper which holds a drop forming apparatus. The appartus consists of a hou6ing having an upper air intake nozzle and an elongate hollow body friction fitted into the stopper. A 10 cc eyringe equipped with a stepping pump is mounted atop the housing with, e.g., a O.01 inch I.D. Teflon coated needle pa~sing through the length of the housing. The interior of the housing iB designed such that the tip of the needle iB subjected to a con~tant laminar air flow which act~ aB an air knife. In u~e, with the syringe full of solution containing the material to be encap~ulated, the stepping pump i8 actuated to incr~mentally force droplets of ~olution from - ~ ) 1 the tip of the needle. Each drop i~ ~cut off" by the air stream and falls approximately 2.5 cm into the CaC12 ~olution where it is immediately gelled by absorption of calcium ions. ~he distance between the tip of the needle and the surface of the CaC12 BolUtion iB great enough, in this instance, to allow the sodium alginate/cell suspension to assume the most physically favorable shape; a sphere (masimum volume for minimum surface area). Air within the tube bleeds through an opening in the stopper. This results in "croas-linking" of the gel and in the formation of a hi~h viscosity shape-retaining protective tem-porary capsule containing the suspended tissue and its mediwm.
The capsules collect in the solution as a separate phase and may be separated by 2spiration.

In the next step of the process, a semipermeable membrane is deposited about the surface of the temporary capsules by "cross-linking" surface layers. This may be effected by subjecting the gelled temporary capsules to an aqueous solution of a polymer containing cationic groups reactive with anionic functionalities in the gel molecules. Polymers containing acid reactive groups such as free imine or amine groups are preferred.
In this situation, the polysaccharide gum $8 cro~slinked by interaction (salt bond formation) between the carbosyl groups and the amine or imine groups. Permeabil$ty can be controlled within limits by selecting the molecular weight of the cross-linking polymer u~ed and by regulating the concentration of the polymer solution and the duration and temperature of esposure. A 801u-tion of polymer having a low molecular weight, in a given time period, will penetrate further into the temporary capsules than ' -13-- (;

1~72586 1 will a high molecular weight polymer. The degree of penetration of the cross-linker has been correlated with the resulting per-meability. In general, the higher the molecular weight and the le6s penetration, the larger the pore size. Broadly, polymers within the molecular weight range of 3,000 to 100,000 daltons or greater may be used, depending on the duration of the reaction, the concentration of the polymer solution, and the degree of per-meability desired. One ~ucce~sful set of reaction conditions, using polylysine of average molecular weight of about 35,00~
daltons, involved reaction for two minutes, with stirring, of a physiological saline solution containing 0.0167 percent polyly-sine. This results in membranes having an upper permeability limit of about 100,000 daltons. Optimal reaction conditions suitable for controlling permeability in a given system can readily be determined empirically in view of the foregoing guide-lines. Using this method it i~ possible to set the upper perme-ability limit of the membranes at a selected level generally below about 150,000 daltons.

Examples of suitable cross-linking polymers include proteins and polypeptides, either natural or ~ynthetic, having free amino or imino groups, polyethyleneamine~, polyethylene-imines, and polyvinylamines. Polylysine, in both the D and L
forms, has been used with euccess. Proteins such a~ polyargi-nine, polycitrulline, or polyornithine are also operable.
Polymers in the higher range of positive charge den~ity, e.g., polyvinylamine, vigorouely adhere to the anionic groupa of the gel molecules to form stable membranes, but the membrane~ are somewhat difficult to di~rupt.

. . .

~3.72586 1 Treatment with a dilute ~olution of gum or a ~wi~-terionic buffer will tie up free amino group~ on the surfaces of the capsules which otherwise may impart to the cap~ule6 a tendency to clump.

At this point in the encapsulation, c~psule~ may be collected which compri~e a semipermeable membrane surrounding a gelled solution of gum, cell-type compatible culture medium, cells, and ~yri~*rLLy an internal matrix of collagen or another - anchorage sub~trate. Since mass transfer should be promoted within the capgules and acros~ the membranes, it i~ preferred to reliquify the gel to its water-soluble form. This may be done by re-establishing the condition~ under which the gum i~ a liquid, e.g., removing the calcium or other multifunctional cation~ from the interior gel. The medium in the capsule can be resolubilized simply by immer~ing the capsule~ in phosphate buffered saline, which contains alkali metal ions and hydrogen ions. Monovalent ions exchange with the calcium or other multifunctional ions within the gum when the capsules are im~er~ed in the solution with stirring. 80dium ci~rate solutions may be u~ed for the ~ame purpose, and serve to ~equester the divalènt ions.

Cell cultures encapsulated as described above may be suspended in growth medium designed specifically to ~atisfy all of the requirements of the particular cell type involved and will continue to undergo normal in vitro metaboli~m and mitosis. If the culture requires an environment of high molecular weight com, ponent~ uch a8 sorum components, tho~e may be omitted from the estrac~p~ular medium. Typically, the component~ normally ingested by cells are of relatively low molecul~r weight and . .

~ 72586 1 readily diffuse across the cap~ule membranes into the microen-vironment of the cells where they permeate the cell me~brane.
Metabolites of the cells which are e~creted into the intracap-sular medium, if they have a molecular weight below the upper limit of permeability of tbe capsule membrane, likewige diffuse thereacros~ and collect in the extracapsular medium.

The encapsulated cells are grown under condition~ of, e.g., temperature, pH, and ionic environment, identical to con-ventional cultures. Cell metabolites may be harve~ted from the extracapsular medium or from the intracapsular volume by conven-tional techniques. However, the culturing technique disclosed herein has the following advantagess 1. The cells of the culture are protected from con-tamination by factors having dimensions in escess of the upper permeability limit of the membranes. This meAns that ~terility requirements normally incident to culturing procedures can be somewhat relaxed, ~ince microorganisms cannot reach the encap-sulated cells.

2. The cap~ules in effect immbbili~e the cells within an environment in which enclosed high molecular weight mAterials are confined, yet lower molecular weight cell nutrient~ and products are readily romoved and introduc~d. Thi8 allowe the - nutrient medium to be intermittently or continuously collected and upplQmontsd a~ desired, without dicturbing the cells.

3. 8ubstance~ of intero~t produced by the cellfi ~re more ea~ily rocovered. Cell products of molecular dimen~ion~
small enough to permeate the cap~ule me~brane~ collect in the 1 extracapsular medium in admixture with nutrients. However, high molecular weight serum components and the like are not released into the extracapsular medium, thus simplifying recovery of a cell product of interest. Cell products of molecular dimensions in excess of the upper permeability limit of the membranes collect within the capsules. These may be recovered in relatively concentrated form by isolating the capsules and subsequently selectively disrupting the membranes using, for example, the technique disclosed hereinafter.

4. The intracapsular volume provides an environment well suited for cell division. Suspension cultures have been observed to undergo mitosis within the capsule. Anchorage depen-dent cells which in normal cultures grow in a two-dimensional monolayer multiply to form an array within the capsule. Such cells use the interior surfaces of the membrane as a sub-strate and/or anchor to the high molecular weight materials set forth above which are disposed within the capsules. This leads to significant increases in cell density as compared with conventional cultures. The ongoing viability of such cell clusters is aided by the fact that the surface area to volume ratios of the capsules can be quite large, and thus all cells have access to required nutrients, oxygen, etc.
In certain situations it is advantageous to selectively disrupt the capsule membranes to release the cells without damage. One notable example is in the production of interfereon (IFN). Cells capable of producing IFN must be subjected to cer-tain viruses or necleic acids in preparation for the IFN produc-tion stage. Also, in several IFN induction procedures, reagents .

, ~72586 1 are added to the culture to inhibit protein synthesis.
Accordingly, the growth stage of the culturing process must be conducted under conditions quite different from the IFN in-duction stage. If the substances used for IFN induction are of a molecular weight in excess of the upper permeability limit of the capsule membranes (as will be the case in virus induc-tions) the induction process cannot be accomplished in the encapsulated cell culture. Accordingly, IFN producing cells, if grown within the capsule, would have to be released by disruption of the membrane in order to be subjected to the induction process.
Disruption of Membranes Cells confined in membranes of the type set forth above may be released by a process involving commercially available reagents having properties which do not significantly adversely affect the encapsulated cells. First, the capsules are separated from their suspending medium, washed thoroughly to remove any contaminants present on the exterior of the microcapsules, and then dispersed, with agitation, in a mixed solution of monatomic, multivalent cations such as calcium ions and a polymer having plural anionic moieties such as a salt of a polysulfonic 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 gum originally employed to form the membranes.
The molecular weight of the polymer should be at least com-parable to and preferably greater than the molecular weight of the polymer having plural cationic groups used in forming the membrane. Within the suspension of capsules in the - ( :L172586 mixed solution, the calcium ions compete w~th the cationic polymer chains used to form the membrane for anioDic ~ites on the water-soluble gum. Simultaneouely, the heparin or ot~er polymer having plural anionic moieties dissolved in the solution coanpetes with the ~Inionic gum in the men~rane for cationic sites on the polymer chains. This result~ in a water-dispersable or pre-ferably water-soluble complex of e.g., polyly~ine and heparin, and in association of the monatomic cations with molecules of the gel.

This step rendere the membrane suseptible to dis~olu-tion upon subsequent expoEure to a ~equestering`agent which completes the disruption proces~ by ~aking up monatomic ions from the gel. Cap~ule membrane debris which remains in the medium, if any, can be easily separated from the calls.

The currently preferred solution for the first stage of the ~elective disruption proce~s canprises 1.1% calcium chloride tw/v) and between 500 to 1,500 units of heparin per milliliter of solution. A volume of microcapsules i~ added to this solution sufficient to constitute between about 20~ and 30% of the total 20 volume of suspension. Calcium chloride and heparin are preferred since both reagents are physiologically compatible with most cells and therefore minimize the possibility of cell damage.
Mixtures of strontium salts or other multiv&lent cations (but not Mg++ ion~) may also be used together with the poly~ulfonic or polyphosphoric acid salts of the type set for~h nbove.

In general, the conc~ntration~ of monatomic ions an anionic polymer used in this step may vary widely. Optimum con-~7ZS86 1 centrations may be readily determined empirically, and depend on exposure time as well as the particular polymer used toform the membranes.
The currently preferred sequestering agent for per-forming the selective disruption is sodium citrate, although other alkali metal citrate salts and alkali metal EDTA salts may also be used. When sodium citxate is employed, the optimum concentration is on the order of 50-60 mM. It is preferred to dissolve the citrate or other sequestering agent in isotonic saline so as to minimize cell damage.

The invention will be further understood from the following non-limiting examples.
Example 1: Human Fibroblasts Human fibroblasts obtained by treating conventional monolayer culture with trypsin and EDTA for 5 minutes at 37 C
in a known manner are suspended in a complete growth medium (CMLR 1969, Connaught Laboratories) supplemented with 40%
~v/v) purified fetal calf serum, 0.8% sodium alginate (Sigma) and 200 ug/ml purified calf skin collagen. The density of the cell suspension is about 1.5 x 10 cells~ml.
Next, a 1,5 percent calcium chloride solution is used to gel droplets formed by using a drop forming apparatus as described above. Droplets on the order of 50-500 microns in diameter leave the tip of the needle and immediately gel upon entering the calcium solution.
After 5 minutes, the supernatant solution is removed by aspiration. The gelled capsules are then transferred to a beaker .~

1 containing 15 ml of a golution comprising one part of a 2% 2 (cyclohexylamino) ethane sulfonic acid buffer solution in 0.6~
NaCl (isotonic, phe8.2) diluted with 20 parts 1% CaC12. After a 3 minute immersion, the capsule3 are wa3hed twice in 1~ CaC12.

The capsules are then transferred to a 32 ml solution comprising 0.005% (w/v) polylysine (average MW 43,000 daltons) in physiological saline. After 3 minutes, the polyly~ine solution is decanted. The resulting capsules, having "permanent" semiper-meable membranes, are then washed twice with 1% CaC12, twice with physiological saline, and mixed with 10 ml of 0.03 percent algi-nic acid fiolution.

The capsules resist clumping, and all cfin be seen to contain fibroblasts. Gel on the interior of the cap~ules i8 reliquified by immersing the capsules in a mixture of saline and citrate buffer (pH-7.4) for 5 minutes. All of the foregoing procedures are conducted at 22--37-C.

Under the micro~cope, these cap~ules are obaerved to comprise a very thin membrane which enclose celle. Molecule~
having a molecular weight up to about one-hundred thousand can traverse ~he membranes.

The resulting capsules are ~uspended in CMLR-1969 supplemented with 10~ fetal calf ~erum. After incubation at 37-C
for 4-5 days, the capsules, if e%amined under the microscope, will be found to contain fibroblasts which have undergone mito~ia and display a classical fibrobla~tic morphology within the micro-capsules.

,. . . .. .. . . .. ...

11725~36 1 The capsule membranes may be disrupted without damaging the cells by allowing a 10 ml portion of the micro-capsule suspension containing about 500-1000 capsules per ml to settle. After aspiration of the medium, the capsules are washed twice with saline. The washed capsules are then mixed with a 3.0 ml aliquot containing 1000 units/ml heparin and 1.1% ¢w/v) CaC12. The suspension is agitated at 37C for 3 minutes, after which the capsules are allowed to settle, the supernatant is aspirated off, and the capsules are washed twice with 3.0 ml of 0.15M NaCl. After aspiration of the second wash solution, the capsules are mixed with 2.0 ml of a mixed solution comprising equal volumes of 110 mM sodium citrate and 0.15M
NaCl (pH=7.4). The mixture is hand vortexed for 1 minute to induce dissolution of the membranes after which cells are washed twice in medium.
The fibroblasts are subjected to an IFN-~ superinduc-tion technique according to the Vilcek procedure. Under a 5%
C2 atmosphere (95% air~, the cell suspension is incubated at 37C for one hour in the presence of 100 ug/ml Poly I-Poly C, a double stranded RNA (known IFN-inducer) available from PL

Biochemicals, Milwaukee, Wisconsin and 50 ug/ml cycloheximide (protein synthesis inhibitor, Calbiochem, La Jolla, California.) After one hour, the suspended cells are washed in medium ~CMLR-1969) containing 50 ug/ml cycloheximide and then resuspended in the same solution for 3 hours at 37C under a 5% CO2 atmosphere.
At the completion of this incubation the washing step is repeated and the cells are resuspended in medium containing 50 ug/ml cycloheximide and 5 ug/ml actimomycin D ~a known RNA synthesis inhibitor, Calbiochem) and incubated for 2 hours at 37C under a 5% CO2 atmosphere. The cells are then washed ~, :1~7Z586 1 twice in medium and suspended in serum-free medium at 37C
for 18-24 hours, during which time the fibroblasts secrete IFN-~, which has a molecular weight on the order of 21,000 daltons and may be harvested from the extracapsular medium.
In one experiment conducted with Poly I-Poly C(5S) (sedimentation value, Poly I and Poly C annealed to form double stranded RNA) 2,500 units of IFN-~ were produced per 105 cells in the culture. An identical yield was obtained in a second run using Poly I-Poly C (12S) (double stranded as purchased).
Other embodiments are within the following claims.

Claims (13)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for culturing anchorage dependent cells, said process comprising the steps of:
A. suspending said cells in a culture medium con-taining an anchorage substrate material and all components (A) needed to maintain viability and to support mitosis of said cells and having a molecular weight in excess of a selected level;
B. encapsulating said cells together with said medium and anchorage substrate material within plural semipermeable membranes having an upper limit of permeability sufficient to preclude traverse of said components (A) and said anchorage substrate material and to allow molecules having a molecular weight below said selected level to traverse said membranes;
C. suspending the product of step B in a culture medium containing all components (B) needed to maintain viability and to support mitosis of said cells and having a molecular weight less than said selected level; and D. allowing said cells to undergo mitosis within said membranes.

2. The process of claim 1 wherein said anchorage substrate is a protein.

3. The process of claim 1 wherein said anchorage substrate is collagen.

4. The process of claim 1 wherein the anchorage substrate is calf skin collagen and is included in said suspension at a concentration between about 10/µg/ml and 1.0 mg/ml.

5. The process of claim 1 comprising the additional step of selectively disrupting said membranes after step D to release said cells.

6. The process of claim 1 wherein said cells comprise fibroblasts.

7. The process of claim 1 wherein said components (A) comprise serum components.

8. The process of claim 1 wherein said cells comprise human fibroblasts capable of secreting interferon.

9. The process of claim 1 wherein said cells comprise fibroblasts capable of secreting interferon, said process com-prising the additional steps of:
E. selectively disrupting said membranes after step D
to release said cells;
F. subjecting said cells to an interferon induction process;
G. incubating the cells resulting from step F in a culture medium; and H. harvesting interferon from the medium of step G.

10. The process of claim 1 wherein during said encap-sulation step spheroidal membranes having an average diameter in the range of 100-500 microns are produced.

11. The process of claim 1 wherein said anchorage substrate comprises a protein having plural free cationic groups.

12. The process of claim 1 wherein said selected level is below about 2.0 x 105 daltons.

13. The process of claim 1 wherein said selected level is less than about 1 x 105 daltons.