ENTRAPMENT OF ANCHORAGE-DEPENDENT CELLS

Abstract: Method and related products for entrapping anchorage-dependent cells and tissues in a permeable gel-like material, nurturing and growing such cells within the gel-like mini-environment while supplying needed nutrients and other materials through the permeable gel from a macro-environment, and harvesting the metabolic and/or other products or by-products.

Description: E TRAPM ENT OF ANCHORAGE-DEPE NDENT- CELLS This application is a continuation- in-part of U.S. Application Serial No. 747 ,977, filed June 24, 1985. F-I EL D OF THE INVENTION The present invention relates to a process for entrapment, preservation and \ or gr owth of anchorage-dependent cells and tissues in an artif icial environment. More particularly, the present invention deals with methods and related products for entrapping anchorage-dependent cells and tissues in a permeable gel-like material, nurturing and growing such cells within the gel-like mini-environment while supplying needed nutrients and other materials through the permeable gel f rom » a macro-environment, and harvesting the metabolic and/or other products or by-products. The present invention permits in v it ro cell culture or growth of anchorage-dependent cells and tissues to high densities, increased yields of biolog ically produ ced products and many other benef its. Similarly, the present invention permits the entrapment and preservation of - anchorage-dependent cells for long periods of time. BACKGROUND O F TH E INVE NTION There are molecules of great investigative, clinical and perhaps cojnmercial value that can best be produced by growing in culture, anchorage-dependent cells that synthesize them. The problem is that it is no simple matter to grow large quantities of anchorage-dependent cells in an artif icial medium. The well-developed technology of industrial microbiology is adapted to the requirements of bacteria, yeasts and molds. Each single cell, encased in a tough cell wall, is an independent metabolic f actory with fairly simple nutritional requirements; for bacteria, glucose and some simple salts will often suffice. Microorganisms grow well floating f ree in a liquid culture medium in tanks with a capacity of as much as 50,000 gallons, resisting damage even when they have proliferated to form a thick suspension and even when the suspension is stirred vigorously with a mechanical agitator. Mammalian cells are different. They are larger than most microorganisms, more f ragile and more complex. The delicate plasma membrane that encloses an animal cell is not encased in a tough cell wall. The mammalian cell' s nutritional requirements are more stringent than those of most microorganisms and indeed have not yet been fully def ined. Rather than being a free-living organism, a mammalian cell is adapted to a specialized lif e as part of an organized tissue, dependent on the specialized functions of many other cells and on a circulatory system that ensures a precisely adjusted and stable env ironment for each cell. Such a cell resists being separated f rom its tissue and grown in an artif icial medium. Most animal cells will not grow at all in suspension; they grow only when they can attach themselves to a surface, thus the name anchorage-dependent. Over the years techniques have been developed for growing anchorage-dependent cells on a small scale in the laboratory. However, it has proved to be much more difficult to grow them efficiently on even a moderately larger scale. Techniques for moderate- and large-scale production of anchorage-dependent animal cells have not changed signif icantly since their development in the early 1960s. Large-scale growth of anchorage- independent cells (suspension cultures) has been achieved by applying the techniques of submerged cultivation of microbial cells. However, the surface requirements of anchorage-dependent cell types has tended to preclude an analogous development. Cur rent techniques for the propagation of anchorage-dependent cells are based on a multiplicity of small-volume, low-productivity reactors, such as roller bottles. Since it is common for a moderate-sized f acility to operate hundr eds of these growth vessels for a single production run, even a simple manipulation such as medium supplementation requires hundreds of ope rations. More complex adjustments requiring multiple operations per bottle, such as cell harvest, compound the problem accordingly. Costs of equipment, space, and manpower are high for this mode of cell production. In an attempt to overcome these problems and to increase process scale and productivity, alternative methods for the . propagation of anchorage-dependent cells have been suggested. These techniques include : plastic bags or tubes, stacked plates, modif ied roller bottles, packed-bed propagator s, artif icial capillaries, microcarriers, and encapsulation. Su ch techniques have been reviewed prev iously by Litw in, Proc. Biochem., 6_:15 (1971) , Maroudous, "New Techniques in Biophy sics and Cell Biology ", R. H. Pain and B .J. Smith, Eds. (Wiley, New York (1973) ) , and Levine et al. "Cell Culture and its Applications", R. Acton, Ed. (Academic, New York (1977) ) . A more recent innovation in the propagation of anchorage-dependent cells is the microcarrier system. The potentially high surface-to-volume ratio (S/V) in a well- mixed microcarrier system allows a single high-productivity vessel to substitute for many low- productivity vessels, reducing the number of operations required per cell, making practical the application of better environmental controls, and providing a homogenous growth env ironment and cell yield. A single reactor vessel also reduces laboratory space and manpower costs. The microcarrier sy stem is not without its problems, however. The potentially high S/V, and hence high cellular productivity, of the system has not been realized due to so-called "toxic effects - ' of the microcarriers on the growth of certain cell types. These effects are manif ested at < • low carrier concentrations (1 g A50/liter) as an initial loss of 50 - to 75% of the cell inoculum, and at high er carrier concentrations (> 2 g A50/liter) as gr eater deg r ees of cell loss and a general suppression of culture growth. Various strategies have been employed to all viate the "toxic effects", including: pretreating the beads with serum or nitrocellulose, increasing cell inocul um, and adding spent culture medium or additi * cs to the growth medium. It has been proposed that the observed A50 "toxicity " may be the result of the aub ptior of certain critical nutrients by the bea.is. Other s have suggested that microenvironmental effects are critical for cell propagation on microcarriers. - Additional disadvantages include: (1) the cells attached to the carrier are exposed to an external environment, and as such subject to collision, shearing, etc. ; (2) recovery of the cells depending on the degree of attachment - strongly attached cells are often damaged or killed upon treatment with trypsin and similar enzymes; (3) growth of cells typically involves the use of DEAE-chloride, a suspected carcinogen which may be deleterious to the cells if there is leach ing ; (4) bridging between microcarriers resulting in mixing problems; and (5) recoverability of the microcarriers for re-use which has proved impractical in industrial applications. Yet another innovation in the propagation of anchorage-dependent cells is microencapsulation. Over the years, there has been considerable interest in the encapsulation or immobilization of living cells. See generally, K. Mosbach, Ed. , Methods in Enzy logy . Vol. 44, Academic Press, New York, 1976; B.J. Abbott, Ann-. Rp . - Ferm. Proc 2 : 91 (1980) ; R.A. Messing, Ann-,- Rpt. Feτ-m. Proc 4 :105 (1980) ; Shovers, et al. U.S. Patent No. 3 ,733,205 (1973) . More recently, efforts have been concentrated in processes for encapsulating tissue and individual cells, particularly mammalian cells, so that they remain viable and in a protected state within a memb r ane which is permeable to the plethora of nutrients and other mater ials required f or normal metabolic f unctions. One such technique is described in U.S. Patent No. 4 ,391 ,909, wherein tissue cells such as Islet of Lange rhans cells are encapsulated within a sph erical semipermeable membrane compr ising a polysaccharide having acidic groups which have been cross-lin ked with acid reactive groups of a crosslinking polymer for permanence of the protective membrane. The semipermeable membrane has a selected limit of per eability of no greater than about 200,000 daltons, so that serum proteins and other high molecular weight materials necessary for growth can be sealed with the living cells within the semipermeable membr ane, while other, smaller molecular weight metabolites and nutrients can traverse the membrane wall and be interchanged with the outside media. The process therein disclosed comprises suspending the tissue to be encapsulated (and the high molecular weight nutrients) in a physiologically compatible medium containing a water soluble substance that can be made insoluble in water (i.e., gelled) , to provide a temporary protective environment for the tissue. The medium containing the tissue is next formed into droplets by forcing the tissue-medium-nutrient suspension th rough a teflon coated hypodermic sy ringe, the tip of which is subjected to laminar air flow which acts as an air knife. See also U.S. Patent No. 4 ,352,883 , wherein the spheres are formed by forcing the materials through a capillary tube into the center of a vortex created by rapidly stirring a solution of Ca+ + cation. The medium, e.g. a polysacch aride gel, is temporarily gelled in a generally spherical shape by contact with the ca±ciuir solution. Thereafter, these "temporary c toules", are provided with permanent poly meric se.iiipermeable membranes at their outer laye r, formed by perma nently Cre ss-linking or polymeriz ing the capsules wi* h polymers containing reactive groups which can react with specif ic constituents of the poly sacchar ides. This technique has most recently been applied to a meth od of . growing anchorage-dependent cells as disclosed in U.S. Patent No. 4,495,288, wherein the cell to be encapsulated is suspended in a medium containing an anchoring substrate material and other high molecular weight components needed to maintain viability and to support mitosis prior to encapsulation. Such complex prior art processes are not without limitations. For instance, with mammalian anchorage-dependent cells, although it has been possible to encapsulate viable and metabolically active cells within hardened semipermeable membranes, promotion of growth therein has not been satisf actory. Moreover, cell densities thus far ach ievable within such membranes has been less than about 10^ cells per illiliter of culture media. Both of these limitations affect the • amount and recove ry of usef ul and desirable cell products produced by the encapsulated material. The ability to grow anchorage-dependent cells to higher cell densities within a protected env ironment (capsule) would provide a means for achieving gr eater output of desirable cell products. A further disadvantage of prior a rt methods of entrapping such cells is the inability to maintain cell viability at desirable high er cell densities. In addition, ^ ~ restricted permeability of th e capsular membrane prevents access of the encapsulated cells to high molecular weight inducer compounds. This restriction necessitates the release of the cells f rom capsules pr ior to induction of product synthesis. The added steps required to release the encapsulated cells may effect cell viability and/or product formation in response to the inducer. SΠMH ARY DP TΠ R - INVENTION In accordance with the present invention, there is provided a novel approach to the entrapment, preservation and/or propagation of anchorage-dependent cells and tissues and to the recovery of products and by-products provided therefrom. More specifically, there is provided methods of entrapping anchorage-dependent cells and tissues within an artificial gel-like environment so as to permit growth of such cells in in vitro tissue culture media to greater than normal cell densities, maintenance of high cell viability and the harvesting of cell products and by-products produced in the entrapped state. The basic approach to the entrapment/preservation and/or propagation of anchorage-dependent cells in accordance with the present invention involves suspending the anchorage-dependent cells in a solution containing an anchoring substrate and a polysaccharide gum such as alkali metal alginate. The suspension is thereafter formed into droplets which are gelled in a calcium chloride solution, washed and grown in culture media to preserve and/or . proliferate anchorage-dependent cells entrapped therein. As noted above, it has been difficult tσ grow anchorage-dependent cells efficiently on even a moderately large s-cale while maintaining greater cell densities and higher cell viabilities. Previous approaches to solving such problems have not been entirely successful, i.e. the toxicoids and other problems of microcarrier systems and the inability of traditional encapsulation techniques to provide desirable cell densities and viability. In this regard, in contrast to the overcoating methods of U.S. Patent Nos. 4,391,909- (Lim) and 4,495,288 (Jarvis) , it is important in practicing the present invention that no semipermeable membrane be formed on the outside of the hydogel beads, either by crosslinking of the hydrogel or by coating with a further polymer, for a number of reasons. Such coatings may interf ere with the free diffu sion into and out of the hydrogel beads. The added steps required to form the semipermeable membr ane will have a negative effect on cell viabilities and make recovery of cells f rom capsules more difficult. Also these "temporary capsules" must be nearly perfect spheres to insu re formation of a non-leaking capsule. The shape ' of the hydrogel bead in practicing the present invention is of less importance and has no direct bearing on the usef ulness of the resultant hydrogel beads. Another advantage of entrapment of anchorage-dependent cells in accordance with the present invention is that it permits recycling and re-use of the cells contained therein, simply by dissolution of the hydrogel, which leaves the cells intact, and f ree f rom any non- cellular materials. This cannot be easily ach ieved with microcar rier sy stems nor with other encapsulation techniques where the cells are enveloped in an insoluble polymer coating. The present invention overcomes such obstacles in that it allows for entrapment, preservation and/or propagation of anchorage-dependent cells at viabilities in excess of 90% and at cell densities where desirable cell products or by-products can be economically harvested for commercial use. The absence of any semipermeable membrane on the outside of the hydrogel bead permits diffusion of molecules greater than or equal to one million daltons in size. This eliminates the need for any additional steps necessary to release gel-entrapped cells prior to induction of product using high molecular weight inducers. Elimination of added steps will improve the subsequent cell viabilities and/or product formation. BRIEF DES CRIPTION -OP -THE- DRAWING S Figure 1 illustrates one apparatus for entrapping anchorage-dependent cells. Figure 2A depicts the growth and viability of entrapped murine epithelial cells designated C127. Figure 2B depicts secretion of hepatitis-B virus surface antigen f rom gel .entrapped murine epithelial cells designated C127. Figure 3- depicts the growth and viability of entrapped murine f ibroblast cells designated SV-3T3. Figure 4. depicts the growth and viability of entrapped human epitheloid carcinoma cells designated HeLa S3. Figure 5 depicts the growth of murine mammary tumor cells in alginate-entrapped gelatin ■ microcarrier cultures. Figure 6 depicts the growth of Chinese hamster ovary cells in alginate-entrapped gelatin microcarrier cultures. DΈTAIL E D DE S CRIPTION OF- TΗ B 'INVENTION The present invention p-.cv^es a novel approach for the entrapment, preservation and/or propagation of anchorage-dependent cells in- - vitro and harvesting products produced thereby. More specif ically, it has now been discovered that anchorage-dependent cells can be entrapped in hydrophilic gels by a process which is much simpler than those previously used; that such entrapped cells can be grown to large cell densities and maintained for substantial periods of time, without the need for an additional selectively permeable membrane sur rounding the entrapped cells; that such entrapped cells can be used to produce high levels of metabolic or other cellular products, such as hormones, vaccines, interf erons; and that, after a suitable period wherein the production of the desired material(s) is maximized, the used, but viable cells, can be recovered for re- use by resolubilizing the hydrophilic gel to release the entrapped cells, followed by re-entrapment using the same procedure, as described above. The process described herein by which anchorage-dependent cells are entrapped and propagated and their products harvested- theref rom typically include the following steps: A. Reagents: (filter sterilized) 1. 1.0% sodium alginate (Kelco- HV) in 0.9% NaCl 2. 0.9% NaCl 3. 1.2% CaCl 2 4. Trypsin- E DTA solution (Flow Labs) 5. 1% E DTA/0.5% NaCl, pH 7.1 6. Complete culture media 7. Vitrogen-100 : (Collagen Corp. Palo Alto, CA) B. Cells: (standard sterii£- technique employed) 1. Anchorage-dependent cell stock s are maintained in 850 cm^ plastic disposable roller -bottles or standard tissue culture flasks under conditions necessary to maximize cell viability (eg. 150 ml complete media per bottle, 37°C incubation at a rotation rate of 0.25 rpm) . C. Eyperi-mental- Protocol (Standard sterile technique is employed throughout) 1. Cells are harvested f rom roller bottle cultures by removing the culture medium and adding 25 ml trypsin-EDTA solution. Roller bottles are then laid on their side and rolled to spread the trypsin- EDTA over the entire area of the cell monolayer. The trypsin-EDTA is then removed and the process repeated once again. The roller bottle is incubated at 37°C and rotated at 0.25 rpm in a conventional roller apparatus. 2. After 10-20 minutes cells will begin to slough-off the surface of the roller bottle. 50 ml complete culture media is added and the roller bottle is tightly capped and agitated to wash the cells from the surface. The suspended cells are then counted in a hemocytometer. Typically a maximum of 1-3 x 10 8 cells/roller bottle is obtained. 3. 1-5 x 10 8 cells (typically 2 x 10 8 ) are then centrifuged at 800 rpm for 5 minutes. The media is aspirated off and the cell pellet is loosened by gently flicking the centrifuge tube. The cells are then resuspended in 20 ml of a collagen solution (Vitrogen-100) which has been neutralized to pH 6.0-7.0 by the addition of 1.0 N NaOH. The final collagen concentration can be 0.1-1.0 mg/ml. Alternatively, collagen may be replaced by histones, fibronectin, poly L-lysine, crosslinked gelatin microcarriers and other microcarrier particles or other such materials or combinations thereof depending on the requirements of the cell being entrapped. If crosslinked gelatin microcarriers are to be used, it is necessary to preincubate cells with the gelatin particles for a period of time sufficient to permit cell attachment. 4. 80 ml of 1.0% Na alginate is then added and the cells are mixed to form an even suspension. The final alginate concentration is 0.8% , although final concentration of 0.6-1.2% can be used. 5. The cell suspension is then delivered to a conventional two phase spray head using a peristaltic pump. Sterile air is also delivered to the spray head at 3.0-4.0 SC FH. The alginate/cell dr oplets are propelled out of the spray head into 0.5-1.0L 1.2% CaCl2 solution to for m shape- retaining gel beads. Flow conditions are adjusted so that the gel beads are left in CaCl2 for no more than 15 minutes. 6. The gel beads are then washed twice with 0.9% NaCl solution and once with complete media. 7. Cultures are best establish ed by resuspending the gel beads in complete culture media to 20-30% beads (v/v) and incubating at 37°C with mixing. Cultur es are ref ed as needed. Preservation of entrapped anchorage-dependent cells is accomplish ed by modify ing the culture media, i.e. reducing the serum and/or glucose concentration to decelerate the growth of the entrapped cells. 8. Cells are counted by washing a 0.5 ml aliquot of beads with 10 volumes of 0.9% NaCl and dissolving the beads in 1.5 ml 1% E DTA/0.5% NaCl, pH 7.1. After a 10-20 min. incubation at room temperature 7.0 ml trypsin- EDTA is added and the sample is incubated at 37°C for 15-30 minutes with occasional shaking. 1.0 ml 0.4% trypan blue solution is added and the cells are counted in a hemocytometer. 9. Metabolic and other cell products may be harvested f rom the media where said products diffuse into the media. Entrapped cells may be released from the hydogel beads for final harvesting by adding 2-5 volumes of EDTA buffer and incubating for 20 minutes at room temperature. Cell agregrates may be dispersed trypsinization. Although the above-outlined steps represent the preferred mode for practicing the present invention, it will be apparent to those skilled in the art that the above-described approach can vary in accordance with techniques known in the art. The hydrophilic gel used for entrapment is preferably an alginate, which is a natural hydrocclloid derived from seaweed, although other hydrophilic materials such as agarose, agar, carrageenan, chitosan, xanthan gum, poly HEMA, and others known in the art can be used to advantage in particular environments. Highly preferred are clarified long-chain sodium alginates, such as Kelco-Gel HV and Kelco-Gel LV, sold by Kelco Company (San Diego) . These are sodium alginates which are fibrous in nature, are supplied at a neutral pH, (typically about 7.2) and contain approximately 80% carbohydrates, 9.4% sodium, 0.2% calciu , 0.01% magnesium, and 0.1% potassium. Kelco-Gel HV has the higher molecular weight, having a Brookfield viscosity of about 400 (1% solution) to about 250 (2% solution) . Of these products, the Kelco Gel HV is highly pref erred. P ref erably, the hydrocolloid is further clarif ied by sequential filtration through filters having pore sizes of 2.5 , 1.2 and 0.6 microns, respectively, and steriliz ed before use by passage th rough a sterile filter having a pore size of 0.45 microns or smaller. The concentration of hydrocolloid in the mixture should range f rom about 0.5 to about 1.4% , pref erably about 0.6 to 1.2% , most preferably about 0.7-0.9% . This is considerably below percentages previously used, and is believed to result in higher porosity of the gel beads to nutrients and other factors. Attempts at making beads below 0.5 mm in diameter have met with difficulty, even with the fairly viscous Kelco Gel HV, and especially with Kelco Gel LV. The particular anchoring substrate used for propagation of anchorage-dependent cells will depend on the requirements of the cell being - entrapped. Exemplary water soluble anchoring substr ates include collagen, a natural protein which is the ch ief constituent of connective tissue in animals,, collagen plus f ibronectin, histόnes, poly L-lysine, gelatin and the like. Water insoluble anchoring substrates (e.g. crosslinked gelatin particles or commercial microcarriers such as dextran and glass particles) may also be used to advantage. The anchoring substrate - solution is pref erably neutralized to a pH between 6.0-7.0 prior to . suspension of anchorage dependent - cells therein. The final concentration of the water soluble anchoring substrate may range between about 0.1-1.0 mg/ml of alginate. Water insoluble anchoring substrates may comprise up to 50% (V/V) of the final bead volume. Pref erably, the micro-environments which contain the anchorage-dependent cells, the hydrophilic gelling agent, the anchoring substrate and various nutrients and accessory materials, are formed into discrete particles, . pref erably generally spherically- shaped particles. Pref erably, the gelled particles are mobile and thus can be arranged for convenient culturing, treatment and product extraction. Thus, for example, the entrapment beads can be arranged, nurtured, or extracted in packed beds, fluidized beds, in stirred containers, in continuous reactors or treatment units, which themselves are known in the art, e.g. similar to those used for treating ion exchange resins, etc. The conditions of treatment, including temperature, pressure, solvent, and physical treatment should be chosen so that the entrapment beads retain their particulate nature. The condition of treatment of the entrapped cells should also be chosen to maintain viability and growth of the cells contained therein. Thus, the entrapped cells shoι ι .! d not be exposed to extremes of temperature, pH, or to toxic chemicals, for amounts of time which would cause l _>_iz of viability of the desired cells. Temperature may range broadly f rom about 5 °C to about 45°C, pref erably between about 15°C and about 40°C. For many- cell sy stems, growth is optimized at temperatures around 37°C. The pH at which the entrapment gels are maintained may also range broadly between about 5 and 9 , pref erably between about 6 and 8. Various steps in treatment of the entrapped cells may require different pH' s, and pH values outside of the broad ranges can often be tolerated by the cells for limited periods of time without deleterious effect. Viability and growth of anchorage-dependent cells normally require, in addition to an anchoring substrate, access to a source of oxygen for respiration, as well as various nutrients, vitamins, amino acids, salts, and other components, known per se for such cell types. Normally some of these nutrients and other factors will be entrapped within the gel bead along with the cells, so that continuous growth for some periods of time can be maintained without further additions of such factors. However, culture of such cells for production of protein s or other metabolites or products require considerable time, and such production is normally optimized by providing the cells with ready access to the required nutrients and other ingredients. Thus, the entrapped cells are pref erably suspended in or otherwise contacted with a fluid containing oxygen, nutrients, vitamins, minerals, etc. , which can diffuse through the hydrophilic gel to the cells and thus maintain viability and growth. It may also be desirable . o include an anchoring substrate in the media to ' optimize attachment and propagation of the entrapped anchorage-dependent cells. Such substrates (e.g. fibronectin) are constituents of serum supplements normally used in cultur e fluids. Figure 1 illustrates one apparatus which may be utilized in entrapping anchorage-dependent cells in accordance with the present invention. The apparatus comprises a controlled source of sterile air, means for admixing the cells to be grown with the anchoring substrate/hydroph ilic „gel-forming material while- such material is in liquid form, means for feeding the sterile air and admixed cells/hydrocolloid to a standard gas/liquid atomizing spray head, and . a reservoir of material which receives and gels the droplets formed by the spray head. Thus, as shown schematically in Figure 1, the apparatus used in the pref erred embodiment comprises a compressor or other source of compressed air 11, an air flow meter 12, an air filter 13, which has an effective pore size of 0.22 nm (micron) or less, so as to sterilize the air used. The sterilized air then proceeds th rough a control valve 14, to a conventional two-phase spray head 15, where it mixes with the liquid cell/hydrocolloid mixture. The liquid cell hydrocolloid mixture is pref erably formed in a tank 17, and is fed to spray head 15 through a pump 16, which is preferably a controlled constant volume, peristaltic pump as is known in the art. In the spray head 15, the liquid is forced out a small diameter (0.006-0.100 mil) cylindr ical top, which is sur rounded by an annular air passageway. The air contacting the droplets formed at the end of the top frees the droplets from the tips. The droplets are then propelled out into the atmosphere in the form of fine spherical droplets. The droplets then contact the liquid in container 18, which contains a divalent cation gelling agent, which gels the liquid droplets, such as a calcium chloride solution, where the hydrocolloid used is sodium alginate. Other divalent cation gelling agents include the other alkaline earth metals (except magnesium) , other divalent metals, and divalent organic cations, such as ethylene disamine. Preferably, tank 17 and container 18 are both stirred during the process at slow speed, in order to keep the solids f rom settling out and to maintain constant concentration. Pref erably, the flow rates of gas and liquid are adj usted so that the size of the particles or droplets formed ranges f rom about 0.4 to about 2 mm in diameter. The flow rates depend to some extent on the viscosity of the liquid hydrocolloid, which in turn depends on the type and concentration of the hydrocolloid used. The provision of from about 0.4 to 2 millimeter particles, pref erably about 0.6-1.5 millimeter particles, permits sufficient diffusion of nutrients and accessory growth factors into the particles to prov ide for cell growth. The spr ay head or noz zle utilized in connection with this invention need not be the modified hypodermic syringes used in previous process. Rather, standard off-the-shelf biphasic spray heads can be utilized to advantage in making the desired beads. Suitable spray heads include those sold by Spray ing Systems, Inc., such as products sold unde r the designations 1/ 8 and JACN, 1/ 8 JACN 1/ 8 JBg. Other suitable noz zles are available in the art. Pref erably, the noz zles used in this invention are beveled at the outside of this tip to form a conical tip, the sides are sloped at 15° or 30° to the longitudinal axis of the top, to direct the air flow at more of an angle to the droplets formed. Such an angle can be simply ground into the liquid tip orif ice. P ref erred inner diamete rs for the liquid spray tip include 0.006 ", 0.010 ", 0.016", and range in size to a maximum of 0.100" with th e smaller siz es pref erred, to produce smaller droplets. The following examples are given to additionally illustrate embodiments of the present invention as it is presently preferred to practice. It will be understood that these examples are illustrative, and that the invention is not to be considered . as restricted thereto except as indicated in the appended claims. EXAMPL E- 1 Entrapment - • -of - - - Genetically Engineered - - Murine Epithelial - Cells 1. Murine epithelial cells (clone C127 derivatives) were grown as monolayer cultures in 850 cm^ plastic roller bottles using media composed of Iscove' s modification of DMEM supplemented with 10% fetal bovine serum (FBS) , 6 mM ' L-glutamine, 50 units penicillin per ml and 50 micrograias streptomycin per ml (complete media) . 150 ml complete media per bottle was used and bottles were maintained at 37°C at a rotation rate of 0.25 rpm. 2. Cells were harvested f rom roller bottles by trypsinization and counted. 3. 1.1 x 10 8 viable cells ' were centrifuged at 800 rpm for 5 min and the cell pellet was resuspended in 6.0 ml Vitrogen-100 (pH6.0) collagen solution. The final concentration of collagen was 0.50 mg/ml sodium alginate. 4. Kelco HV sodium alginat e was added to a final concentration of 0.8% sodium alginate (i.e. -24 ml of 1% HV sodium alginate) . The final concentration of cells was 3.67 x 10 > cells/ml alginate. 5. Hydrogel/cell beads were delivered at 10 ml min to a two-phase spray head (1650 head, 64SS air cap) with an air flow of 3.0 SC FH. 6. Hydrogel/cell beads were gelled in 0.50L 1.3% CaCl2 , washed tw ice with normal saline and once with complete media. 7. Cultures were established at ratio of 20: 80 [beads: complete medial in a T-flask and incubated at 37°C in a humidified atmosphere containing 5% CO2. 8. Cultures were fed as needed by replacing 50% of the spent culture fluid with fresh complete media. The spent media was stored at - 20°C until assayed for antigen. Antigen was measur ed by radioimmunoassay. 9. Entrapped cells were counted by dissolving 1.0 ml of washed beads in 9 ml 1% E DTA/0.5% NaCl, centrif uging the released cells at 800 rpm for 5 min and resuspending the cell pellet in 4.5 ml trypsin- EDTA solution. 10. After 20-30 min at 37°C, 0.5 ml 0.4% trypan blue solution was added and the., cells counted in a hemocytometer. The growth, viability and ancigen production of entrapped murine epithelial cells (C127) over a two week period is illustrated in Figs. 2A and 2B with and without the use of Vitrogen-100 as. -the anchoring substrate. EX-AMPL E- -II E-n ra -menfr of -Murine -Fjbroblas - ils 1. Murine fibroblast cells (clone SV- 3T3 ; ATC C CCL 163.1) were grown as monolayer cultures in media composed of DME M supplemented with 10% FBS, 50 units penicillin/ml and 50 microgr ams .. streptomycin/ml (complete media) . 2. Fibroblasts were harvested f rom bottles by trypsinization, counted and 6 x 10^ viable cells were centr if uged. 3. Cell pellet .was resuspended in 6.0 ml histone II-A calf thymus (Img/ml) (pH 7.0) and mixed with 24 ml 1% Kelco HV sodium alginate. Final concentration of histone was 0.20 mg/ml alginate. 4. All subsequent steps were as described in Example I, steps 5-10, with the exception of the RIA quantitation of antigen in media. The growth and viability of entrapped mur ine fibroblast cells (SV-3T3) is illustrated in Fig. 3. EX-AMPL E- -III Entrapment -of- -Hu an - Epitheloid -Carcinoma- Ceils 1. Human epitheloid carcinoma cells (HeLa S3 ; ATCC CCL 2.2) were grown as monolayer cultures in T-flasks in media composed of DM EM supplemented with 10% FBS, 50 units pennicillin/ml and 50 micrograms streptomycin/ml. 2. Cells were harvested by trypsinization, counted and 1.2 x 10 8 viable cells were centrifuged. 3. The cell pellet was resuspended in 60ml 0.8% sodium alginate (HV) and further processed as described in Example I, steps 5-10 with the exception of the RIA quantitation of antigen in media. The growth and viability of entrapped human epitheloid carcinoma cells (HeLa S3) is illustrated in Fig. 4. EXAMPL E- IV Entrapment of Murine -Mammary- Tumor -cells- in Aiqjnate-entrapped- -Gela in- tt-i rp-gar-rie g 1. Mouse mammary tumor cells were maintained in 850cm^ sterile disposable roller bottles in media composed of Iscove' s modif ied DM EM (IM) plus 10% fetal bovine serum ( FBS) , 6 mM L-glutamine, 50 units penicillin/ml and 50 meg. streptomycin/ml (complete IM) . Cell passages were carried out by incubation of monolayers with trypsin- EDTA solution. 2. Gelatin microcarriers (K.C. Biological, Lenexa, Kansas, catalogue #M C-540) were prepared as described in the manufactures Procedures Bulletin #38. Gelatin microcarriers were swollen and hydrated overnight in phosphate buffered saline (PBS, pH 7.4, Ca 2+ , Mg 2 + free) . The microcarriers were then washed twice in PBS and mixed with 1 vol. PBS. Sterilization was by autoclaving for 15-30 min. at 120°C, 15 psi. Microcarriers were stored at 4°C in the dark until time of use. Prior to use, the microcarriers were washed overnight in complete media. Alternatively, gelatin microcarries may be prepared in accordance with the protocol set forth in Example VI below. Mouse mammary tumor cells were trypsinized, washed in complete media and counted. Cells were preincubated overnight with 30 ml microcarriers (0.5-2.0 x 10^ cells/ml settled microcarriers) in order to allow for cell attachment. After 15-18 hours at 37°C the culture was divided into 2 equal aliquots and centrifuged. One pellet was resuspended in 125 ml complete media and used as unentrapped control culture. The other was entrapped as described hereinbelow. 3. Microcarriers were centrifuged at 800rpm for 5 min. at room temperature and the supernatant was discarded. The pellet was resuspended in 1-3 volumes of sterile 0.8% sodium alginate and the mixture was entrapped as previously described using a 20/100 spraying head. Microcarrier/ alginate droplets were dropped into a pre- warmed solution of 1.2% calcium chloride. Alginate gel beads were then washed twice in sterile saline and once in complete IM. Alginate gel beads were added to 3 volumes of complete IM in a spinner flask and incubated at 37°C with gentle stirring. Cultures were fed as needed. 4. In preparation for periodic sampling, alginate gel beads and/or unentrapped microcarrier control cultures were resuspended in 10 volumes 1% EDTA/0.5% NaCl and incubated at room temperature until the alginate resolubilization was complete. Samples were spun at 1000 rpm for 5 min. and supernatants were discarded. Pellets were resuspended up to 9 ml in trypsin-EDTA and incubated at 37°C until gelatin microcarriers were completely solubilized. One milliliter 0.1% trypan blue was added and cells counted in a hemocytometer. The growth of alginate-entrapped gelatin microcarrier cultures of mur ine mammary tumor cells is illustrated in Fig. 5. E-X-A L E- V Entrap- ent - of - Chinese- Hamster -Ovary - <CHO) - Cells- in Aiσinate-Entr apped Gelatin- -Micrσcarrier-s. Genetically engineered CH0 cells were seeded onto 10ml of gelatin microcarriers (2x10^ cells/ml gelatin) in a total culture volume of 125 ml . After 24 hours at 37°C the culture was divided into cont r ol (unentrapped) and experimental cultures (entrapped) . Gelatin microcarriers were mixed with 3 volumes of 1% alginate and entrapped as described in Example IV. The growth of alginate-entrapped gelatin microcarrier culture of CH0- cells is illustrated in Fig. 6 as compared with unentrapped microcarrier control cultures. E-X-ftMP£ E- Vi Pr-oduction- of- Crosslinked- Gelatin- Particles I. Reagents 1. gelatin-Type A porcine, 225 Bloom 2. glutaraldehyde-25% solution 3. distilled/deionized water 4. phosphate buffered saline, pH 7.4 (PBS Ca 2 + , Mg 2 + -free) . II. Protocol 1. 100 gm gelatin was slowly added to 800 ml of rapidly mixing water preheated to 60-70°C. When all the gelatin was in solution the volume was adjusted to 1000 ml with water. 2. 20 ml glutaraldehyde was quickly added to the • rapidly mixing gelatin solution. Gelation was complete within 5 min. 3. The crosslinked gelatin was broken into large pieces, rinsed 3 times with 2 volumes of water and then mixed with 1 volume of water. 4. The 50% suspension was transferred to a kitchen blender and liquefied for 30 sec. 5. Gelatin particles were washed 5 times with water " by centrifuging the gel slurry for 5 min. at 3000 rpm and resuspended the pellet in 3-5 volumes of fresh water. 6. The gel particles were then resuspended in 2-3 volumes of a 0.1% gelatin solution and mixed overnight at room temp. 7. Gel particles were then centrifuged as above and washed 3 times in 2-3 volumes of PBS. 8. Gelatin particles were then resuspended in one volume PBS and transferred to storage bottles. 9. Gelatin particles were sterilized by autoclaving for 30 min. at 120°C. 10. Crosslinked gelatin particles were stored in the dark at 4°C and washed overnight with complete media just prior to use.

Claims: wftat- -is- laime - is; 1. A process for propagating anchorage-dependent cells, said process comprising the steps of : a) suspending said cells in an anchoring substrate/alkali metal alginate solution; b) forming said suspension into droplets; c) gelling said droplets to form shape- retaining structures about said anchorage-dependent cells; d) placing said anchorage-dependent cell containing structures in a growth medium which promotes- propagation of anchorage-dependent cells; and e) g rowing said anchorage-dependent cells within said structures. 2. The process of claim 1 , wherein the anchoring substrate is selected from the group of collagen, fibronectin, histones, poly-L-lysine, microcarriers or mixtures thereof . 3. The process of claim 2, wherein the concentration of the anchoring substrate selected f rom the group of collagen, fibronection, histones, poly-L-lysine or mixtures thereof is f rom about 0.1 to about 1.0 mg/ml alkali metal alginate solution. 4. The process of claim 2, wherein the microcarriers comprise upto about 50% of the volume of the shape- r etaining structures. 5. The process of claim 2, wherein the microcarriers comprise particulate material selected from the group of crosslinked gelatin, dextran or - glass. 6. The process of claim 1, wherein the concentration of said alkali metal alginate component of said solution is from about 0.6% to about 1.2% w/v dissolved in physiological saline. 7. The process of claim 6, wherein said alkali metal alginate is sodium alginate. 8. The process of claim 1, wherein said droplets are gelled by contacting said droplets with a gelling solution containing f rom about 0.6 to about 1.5% w/v of isotonic calcium chloride. 9. The process of claim 1, wherein said gelled droplets range in size from about 0.5 mm to about 2.0 mm in diameter. 10. The process of claim 1, wherein said anchorage-dependent cells proliferate to cell densities greater than about 5 x 10^ cells/ml of culture medium. 11. The process of claim 1, wherein said anchorage-dependent cells are selected from the group of transformed animal cells of a species type which can either naturally or by means of genetic engineering be made to produce and secrete products of biological/commercial importance. 12. A process for producing a substance which is produced by anchorage-dependent cells, said process comprising: a) suspending said cells in an anchoring substrate/ alkali metal alginate solution; b) forming said suspension into droplets; c) gelling said droplets to form shape- retaining structures about said anchorage-dependent cells; d) placing said anchorage-dependent cell containing structures in a growth medium which promotes propagation of anchorage-dependent cells; e) allowing said cells to undergo metabolism in- vitro to produce said substance; and f) harvesting said substance f rom the growth medium. 13. The process of claim 12, wherein the anchoring substrate is selected f rom the group of collagen, fibronectin, histones, poly-L-lysine, microcarriers or mixtures thereof . 14. The process of claim 13, wherein the microcarriers comprise particulate material selected from the g roup of crosslinked gelatin, dextran or glass. 15. The process of claim 12, wherein said alkali metal alginate is sodium alginate and said droplets are gelled by contacting said droplets with a calcium chloride solution. 16. The process of claim 15, wherein said substance diffuses into and is harvested f rom said growth medium. 17. The process of claim 15, including the additional step of releasing said cells f rom said shape- retaining structures after harvesting said substance. 18. The process of claim 15, wherein said cells are released from said structure by addition of an EDTA buffer followed by incubation at room temperature. 19. A process for prese rving anchorage-dependent cells, said process comprising the steps of : a) suspending said cells in an anchoring substrate/alkali metal alginate solution; b) forming said suspension into droplets; c) gelling said droplets to form shape- retaining structures about said anchorage-dependent cells; d) placing said anchorage-dependent cell containing structures in a growth medium which promotes maintenance of anchorage-dependent cells; and e) maintaining said anchorage- ' pendent cells within said structures. 20. The process of claim 19, wherein the anchoring substrate is selected from the group of collagen, fibronectin, histones, poly-L-lysine, microcarriers or mixtures thereof . 21. The process of claim 20, wherein the microcarriers comprise particulate material selected from the group of crosslinked gelatin, dextran or glass. 22. The process of claim 19, wherein said alkali metal alginate is sodium alginate and said droplets are gelled by contacting said droplets with a calcium chloride solution.

Inventors: BUEHLER ROBERT, SINACORE MARTIN, VASINGTON PAUL, BUEHLER, ROBERT, SINACORE, MARTIN, VASINGTON, PAUL
Applicants: KARYON TECHNOLOGY INC, KARYON TECHNOLOGY, INCORPORATED
Application year: 1986
Main classification: C12N5
IPC codes: C12N5