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Publication numberWO1985005630 A1
Publication typeApplication
Application numberPCT/US1985/000838
Publication date19 Dec 1985
Filing date8 May 1985
Priority date1 Jun 1984
Also published asEP0185701A1, EP0185701A4
Publication numberPCT/1985/838, PCT/US/1985/000838, PCT/US/1985/00838, PCT/US/85/000838, PCT/US/85/00838, PCT/US1985/000838, PCT/US1985/00838, PCT/US1985000838, PCT/US198500838, PCT/US85/000838, PCT/US85/00838, PCT/US85000838, PCT/US8500838, WO 1985/005630 A1, WO 1985005630 A1, WO 1985005630A1, WO 8505630 A1, WO 8505630A1, WO-A1-1985005630, WO-A1-8505630, WO1985/005630A1, WO1985005630 A1, WO1985005630A1, WO8505630 A1, WO8505630A1
InventorsPaul John Vasington, Maurice Mark Lynch, Maureen Elizabeth Frye
ApplicantKaryon Technology, Incorporated
Export CitationBiBTeX, EndNote, RefMan
External Links: Patentscope, Espacenet
Tissue culture and production in permeable gels
WO 1985005630 A1
Methods and related products for entrapping living biological materials such as tissues and cells 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.
Claims  (OCR text may contain errors)
What is Claimed is:
1. A process for proliferating viable mammalian cells, said process comprising: a) suspending said cells in an alkali metal alginate solution; b) forming the suspension into droplets; c) gelling said droplets to form shape- retaining structures about said cells; d) placing said cell containing structures in a growth medium which promotes proliferation of said cells; and e) growing said cells within said structures.
2. The process of claim 1, wherein said alkali metal alginate solution is from about 0.6 - 2.0% w/v dissolved in physiological saline.
3. The process of claim 2, wherein said alkali metal alginate is sodium alginate.
4. The process of claim 1, wherein said droplets are gelled by contacting said droplets with multivalent cation gelling agent.
5. The process of claim 4, wherein said multivalent cation gelling agent is a solution containing about 0.6 - 1.5% w/v of calcium chloride.
6. The process of claim 1, wherein said gelled droplets range in size from about 0.5 mm to about 2 mm in diameter. 7. The process of claim 1, wherein said mammalian cells proliferate to cell densities greater than about 5 x 106 cells/ml of culture medium.
8. The process of claim 1, wherein said cells are hybridoma cells.
9. The process of claim 8, wherein said hybridoma cells proliferate to cell densities greater than about 5 x 106 cells/ml of culture medium.
10. The process of claim 1, wherein said cells have been genetically modified.
11. A method of preserving living mammalian blood cells, comprising: a) suspending said cells in an alkali metal alginate solution; b) forming the suspension into droplets; c) gelling the droplets to form shape- retaining structures about said cells; d) placing the cell containing structures in a growth medium which promotes maintenance of the cells; and e) maintaining the cells w ithin the structures.
12. A process for producing a substance which is produced by viable cells, said process comprising: a) suspending said cells in an akali metal alginate solution; b) forming said suspension into droplets;
SUBSTITUTE SHEET c) gelling said droplets to form a shape- retaining semipermeable gel about said cells; d) placing said gel- entrapped cells in a growth medium which promotes prolifera tion of said cells; e) allowing said cells to undergo metabolism in vitro and to produce said substance; and f) harvesting said substance.
13. The method of claim 12, wherein said cells comprise mammalian cells.
14. The method of claim 12, wherein said cells comprise hybridoma cells.
15. The method 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 method of claim 12, wherein said substance diffuses into and is harvested from said growth medium.
18. The process of claim 12, wherein said cells have been genetically modified.
19. The process of claim 12, wherein said gelled droplets range in size from about 0.6 mm to about 2 mm in diameter.
20. The entrapped cells of claim 12.
Description  (OCR text may contain errors)


Field of the Invention

The present invention relates to a process for entrapment and growing cells and tissues in an artif icial environment. More particularly, the present invention deals with methods and related products for entrapping living biological materials such as tissues and cells 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. The present invention permits in vitro cell culture and growth to high cell densities, increased yields of biologically produced products and many other benefits.

Background of the Invention

Over the years, there has been considerable interest in the encapsulation or immobilization of living cells, particularly those of microbial origin. See generally, K. Mosbach, Ed., Methods in Enzymology, Vol. 44, Academic Press, New York, 1976; B.J. Abbott, Ann. Rpt. Fer . Proc . 2: 91 (1980) ; R.A. Messing, ______

Rpt. Fer . Proc . 4:105 (1980) ; Shovers, et al. U.S. Patent No. 3,733,205 (1973) . Interest has been extended to the immobilization of plant cells in suspension. P. Brodelius et al., FEB S Letters. 103. 93-97 (1979) .

More recently, efforts have been concentrated in processes for encapsulating tissue and individual cells, particularly mammalian cells, so that they

* *-*<'_,? . n__*--p_?v - s-TS>^ _"T< __- w SSIπ--! P-PcT i remain viable and in a protected state within a membrane which is permeable to the plethora of nutrients and other materials required for normal metabolic functions.

One such technique is described in U.S. Patent No. 4,391,909 (Lim) , wherein tissue cells such as Islet of Langerhans cells are encapsulated within a spherical semiper eable membrane comprising a pol saccharide having acidic groups which have been cross-linked for permanance of the protective membrane. The semipermeable membrane has a selected limit of permeability 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 membrane, 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 through a teflon coated hypodermic syringe, 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 polysaccharide gel, is temporarily gelled in a generally spherical shape

SUBSTITUTE SHEET by contact with the calcium solution. Thereafter, these "temporary capsules" are provided with permanant polymeric semipermeable membranes at their outer layer, formed by permanently cross-linking or polymerizing the capsules with polymers containing reactive groups which can react with specific constituents of the polysaccharide.

Thus until the present invention, entrapment in alginate gels alone was considered as only a "temporary" vehicle, around which a permanent membrane could be formed. Generally, following the formation of the permanent membrane, the "temporary" gel was dissolved, so that any cell growth achieved thereafter was not in the presence of the gelled substance.

Such complex prior art processes are not without limitations. For instance, with mammalian cells, although it has been possible to encapsulate viable cells within hardened semipermeable membranes, promotion of growth therein has not been satisfactory. Moreover, cell densities thus far achievable within such membranes has been less than about 106 viable cells per milliliter of culture media. Both of these limitations affect the amount and recovery of useful and desirable cell products produced by the entrapped material. The ability to grow cells to higher cell densities within a protected environment (capsule) would provide a means for achieving greater output of desirable cell products.

A further disadvantage of prior art methods of entrapping animal cells is the inability to maintain cell viability at desirable higher cell densities. See P. Brodelius et al., FEBS Letters, supra, where

SUBSTITUTE SHEET entrapment of mammalian cells resulted in a lack of proliferation of cells and a cell viability of about 10-30% after incubation in tissue culture for one (1) week.

Summary of the Invention

In one aspect, the present invention provides a process for proliferating cells within an artificial gel-like environment. The methods and products involved permit the growth of mammalian cells in ___. vitro tissue culture media to greater than normal (unentrapped) cell densities, the maintenance of high cell viability in such material, and the collection of by-products produced as a result of entrapped cell metabolism.

The basic approach involves suspending the cells in a polysaccharide gum, preferably an alkali metal alginate such as sodium alginate and thereafter forming the suspension into droplets. The_ droplets thus formed are gelled in a calcium chloride solution, washed and grown in culture media to proliferate cells entrapped therein.

More specifically, the present invention provides a process for proliferating viable mammalian cells (including hybridoma cells) within a semipermeable gel-like membrane. As noted above, it has previously been difficult to maintain viable mammalian cells in an artificial environment at levels greater than 10-30% viability. It has also been extremely difficult to grow mammalian cells in artificial environments, i.e. capsules, particularly at cell densities where commercial quantities of cell products are produced. The present invention overcomes such obstacles in that it allows entrapment of viable mammalian cells at viabilities exceeding 50% and at cell densities where desirable cell products can be economically harvested for commercial use.

In another aspect of the present invention, there is provided a process for producing substances which are produced by viable cells which comprises the above-described entrapment technique.

Brief Description of the Drawing

Figure 1 shows a schematic depiction of one embodiment of various apparatus which are usef ul in connection with the present invention.

Figure 2 depicts the growth, viability and antibody production of entrapped v. non-entrapped murine hybridoma cells designated KTI-2A.

Figure 3 depicts the growth, viability and antibody production of entrapped v. non-entrapped human B-cells designated KTI-7A. _

Detailed D scri i on of the Invention

It has now been discovered that mammalian and other living cells can be entrapped in hydrophilic gels by a method and using apparatus 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 surrounding the entrapped cells; that such entrapped cells can be used to produce high levels of metabolic or other cellular products, such as monoclonal antibodies; and that, after a suitable

SUBSTITUTE SHEET 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 present invention is particularly well suited for the production of monoclonal antibodies using hybridomas entrapped in a hydrophilic gel. However, other cell types, particularly mammalian can also be used to advantage to produce other products in accordance with the present invention. Other types of cells which can be used to produce desired products include various types of T-cells, e.g. helper T-cells, suppressor T-cells, B-cells, mast and stem cells, hormone-producing cells from the pituitary or other glands or tissues in the body; and other tissue which produces or can be modif ied to produce the product of interest.

The hydrophilic gel used for entrapment is preferably an alginate, which is a natural hydrocolloid derived from seaweed, although other hydrophilic materials such as agarose, agar, carrageenan, chitosan, xanthan gum, polyacrylamides, poly HEMA, and others known in the art can be used to advantage in particular environments.

Preferably, the micro-environments which contain the cells, the hydrophilic gelling agent and various nutrients and accessory materials, are formed into discrete particles, preferably generally spherically- shaped particles. Preferably, the gelled particles are mobile and thus can be arranged for convenient culturing, treatment and product extraction. Thus,

SUBSTITUTE SHEET 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 beads should also be chosen to maintain viability and growth of the cells contained within the beads. Thus, the beads should not be exposed to extremes of temperature pH, or to toxic chemicals, for amounts of time which would cause loss of viability of the desired cells. Temperature may range broadly from about 5C to about 45C, preferably between about 15C and about 40C. For many cell systems, growth is optimized at temperatures around 37C. The pH at which the entrapment gels are maintained may also range broadly between about 5 and 9, preferably between about 6 and 8. Various steps in treatment of the beads 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.

Cell viability and growth normally require access to a source of oxygen for respiration, as well as various nutrients, vitamins, amino acids, salts, and other components, known per se for various cell types. Normally some of these nutrients and other factors will be entrapped within the bed along with the cells, so that continuous growth for some periods of time can

SUBSTITUTE SHEET be maintained without further additions of such factors. However, culture of such cells for production of proteins 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 beads are preferably 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.

As shown in Figure 1, one apparatus utilized in entrapping cells in accordance with the present invention involves a controlled source of sterile air, means for admixing the cells to be grown with the hydrophilic 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 preferred 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 im (micron) or less, so as to sterilize the air used. The sterilized air then proceeds through 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 preferably 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 - 0.016 mil) cylindrical top, which is surrounded 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 from settling out and to maintain constant concentration.

As noted above, previous attempts to grow mammalian cells in. hydrogels, particularly without a porous outer semipermeable membrane, have met with little or no growth rate and poor viability. It has now been found that, by controlling particle size and the type of hydrogel used, mammalian cells can be entrapped or encapsulated and grown to high densities, with substantially improved viability of cells.

SUBSTITUTE SHEET One important factor is the type of hydrocolloid to be used. 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% calcium, 0.01% magnesium, and 0.1% potassium. Kelco- Gel HV has the higher molecular weight, having a Brookfield viscosity of about 400 (1% water solution) to about 3500(20%) water solution) centipoise wherein Kelco Gel LV has a viscosity of about 50 (1% solution) to about 250 (2% solution). Of these products, the Kelco Gel HV is highly preferred.

Preferably, the hydrocolloid is further clarified and sterilized before use by passage through a sterile filter having a pore size of 0.45 microns or smaller.

Preferably, the flow rates of gas and liquid are adjusted so that the size of the particles or droplets formed ranges from about 0.4 to about 2mra 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, preferably about 0.6 - 1.5 millimeter particles, permits sufficient diffusion of nutrients and accessory growth factors into the particles to provide for cell growth. Substantially larger gel particles may decrease the growth and viability rates of the cells. The concentration of hydrocolloid in the mixture should range from 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 0.5mm in diameter have met with difficulty, even with the fairly viscous Kelco Gel HV, and especially with Kelco Gel LV.

A key feature is to achieve cell- containing hydrogel beads with sufficient porosity and an appropriate size for diffusion of the nutrients to the cells in the inner reaches of the beads. While the Kelco Company products mentioned have been utilized in overcoming the problems of the prior art and growing mammalian cells in an encapsulated environment, many similar or alternative hydrogels exist in the art and are commercially available.

It having been shown that improved growth and viability rates can be obtained from such materials without use of the overcoating method of U. S. Patent No. 4,391,909 (Lim) those skilled in the art can adj ust the process to other similar materials. It is important that no semipermeable layer be formed on the outside of the hydrogel cell beads, either by cross linking of the hydrogel or by coating with a further polymer, for a number of reasons. Such coatings may interfere with the free diffusion into and out of the hydrogel beads. Moreover, the hydrogel beads of the present invention permit recycling and re-use of the cells contained therein, simply by dissolution of the hydrogel, which leaves the cells intact, and f ree from any non-cellular materials. This could not be done if

SUBSTITUTE SHEET the cells are enveloped in an insoluble polymer coating.

The spray head or nozzle utilized in connection with this invention need not be the modified hypodermic syringes used in previous products. 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 Spraying Systems, Inc., such as products sold under the designations 1/8 and JACN, 1/8 JACN 1/8 JBg. Other suitable nozzles are available in the art. Preferably, the nozzles 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 orifice. Preferred inner diameters for the liquid spray tip include 0.006", 0.010" and 0.016", with the smaller sizes preferred, to produce smaller droplets.

One typical example of gel entrapment includes the steps of suspending cells at a concentration of about 106 cells/ ml in a 1.5% (w/v) solution of sodium alginate in normal saline. This suspension is placed in a suitably- sized vessel. Where the production run is to take considerable time, so that the cells will be out of contact with media for considerable time, nutrients and other materials can be added to the alginate suspension and/or to the multivalent gelling agent solution. A typical addition would include 50 mm of glucose, IX of essential amino acids, IX of nonessential amino acids, IX of vitamins and/or any other needed growth factors. A tube from this vessel

SUBSTITUTE SHEET is connected to the liquid inlet of the spray head appartus. Another tube containing compressed air is connected to the air inlet of the spray head apparatus. The liq uid is pumped through the spray head at the same time compressed air is blown through the spray head. The resultant sodium alginate cell suspension droplets are blown into the gelling solution of calcium chloride (1.0 - 1.3% (w/v) ) . The contact with calcium ions causes the immediate formation of a gel (calcium alginate) which entraps the cells contained within the gel droplet. Upon complete formation of all droplets, the droplets are removed from the calcium chloride solution, washed several times in normal saline solution and placed in the appropriate tissue culture medium. Entrapped cells have been shown to divide and metabolize for several weeks in these permanent calcium alginate gels. Cells are capable of attaining higher cell densities than if grown in normal tissue culture. The cell viabilities at these higher densities has also been shown to remain high 050%) .

In a preferred general process similar to that just described, seed cells for entrapment are cultured in conventional stir red-tank reactors as suspension cultures, typically in Iscove's Modif ied Dulbecco's Medium containing 5-10% (vol/vol) fetal bovine serum and 6mM L-glutamine. Cells are harvested in the logarithmic phase of growth, at viabilities greater than or equal to 90% . All steps in the entrapment process are performed in laminar-flow cabinets providing H EPA- iltered air. Harvesting is done by pouring cell suspensions into sterile 800-ml glass centrifuge bottles and centrifuging for 10-15 minutes at 1500 rpm in an IE C CRU-5000 centrifuge to pellet the cells. All but 20-25 ml of the supernatant culture medium is removed by aspiration, and the cell pellets are gently resuspended in the remaining fluid. Sterile physiological saline and 1.0% (wt/vol) sodium alginate are then added to the pooled cells to effect a final alginate concentration of 0.8% (wt/vol) , and a cell density of 2xl06 cells per ml. The alginate-cell suspension is then extruded through a manifold droplet-forming apparatus (Spraying Systems 8490-SS) outfitted with four 1650 SS liquid nozzles modified as outlined above, 64SS air caps, and needle valves to control liquid flow through the individual nozzles. The alginate-cell suspension is pumped through the manifold using a peristaltic pump (Cole-Parmer) and an airflow of 5 SCFH. Compressed air from a tank or in- house system is used, and sterilized by passage through a 0.22-micron hydrophobic filter before contacting the manifold. Droplet formation rate is controlled by pump speed and is typically on the order of 28-30 ml per minute per manifold. The alginate droplets thus formed fall into a receiving bath of sterile isotonic gelling solution composed of 1.3% calcium chloride dihydrate, 0.5% glucose, and 13 mM HEPES adjusted to pH 7.4. Exposure of the alginate droplets to the gelling solution causes the displacement of Na+ ions f rom the alginate by Ca++ ions and the formation of calcium alginate spheres, or beads, of 0.2-1.0 mm diameter. The alginate beads are transferred to a wash vessel and allowed to settle out of suspension. The supernatant gelling solution is aspirated from the beads and replaced with four times the settled bead volume of sterile physiological saline. This washing process is repeated three times; once more with sterile physiological saline, and twice with Iscove's Modif ied Dulbecco's Medium without Serum or L-glutamine. The last wash solution is removed and the beads resuspended in Iscove's Modified Dulbecco's Medium supplemented with 5-10% fetal bovine serum, 6mM L-glutamine, and 50 ug/ml of gentamicin sulfate.

By-products from cell metabolism have been collected f rom gel-entrapped cell cultures produced by the above- described processes. For example, hybridoma cells (i.e. cells produced as a result of fusing spleen cells or antibody producing cells with a myeloma cell line either intra- or interspecies) may be entrapped in a calcium alginate gel-like material. These hybridoma cells may be obtained commercially, e.g. from the American Type Culture Collection, Rockville, MD, or may be prepared by any individual skilled in the art of tissue culture, immunology and hybridoma development. See ATCC Catalog, Cell Lines. Viruses. Antisera. 192 et seq (ATCC 1983) ; Kohler and Milstein, Nature 256: 495 (1975) , the disclosure of which is incorporated herein by reference. Each individual hybridoma cell line may have its own unique set of growth requirements, i.e. type of tissue culture media and type and amount of nutrients req uired, as is recognized by individuals skilled in the art.

Under normal in vitro tissue culture conditions, most hybridoma cell lines grow to densities of 105 - 2X106 cells/ml of tissue culture media. These cells typically produce monoclonal antibodies in vitro at levels of 1-10 jig/ml/day of culture media depending on the cell line. Growth of hybridoma cells to higher cell densities, as attained in the present invention, effectively increases the yield of monoclonal antibody/ ml of culture media resulting in significant space and cost advantages. Entrapment of hybridoma cells in calcium alginate provides the means by which cell densities can be increased above 2X106 cell/ml and for production of monoclonal antibody at levels above the normal 1-10 jig/ml. One procedure achieving such production is described as follows:

Using aseptic procedures, cells from a particular hybridoma cell line are separated f rom their culture fluid by low speed centrifugation (500 x G) in sterile conical test tubes. The supernatant is removed and the cells are suspended to a concentration of 1X105

2X106 cells/ml in a sodium alginate solution (e.g., Kelco Gel HV) at a concentration of 0.5 - 2.0% , preferably 0.6 - 1.5% , preferably in normal saline.

All work is carried out using aseptic techniques in a laminar flow hood. Air pressure is adjusted to 0.10 SCFH (standard cubic feet per hour) using the air flow meter 12 (e.g. Dwyer Flow Master #SS-2MHL-25) . All equipment and tubing which the alginate cell suspension passes through has been sterilized. A 0.22 iim in-line air filter (e.g. Millipore Millex-GS) sterilizes the air prior to its passage through the spray head assembly.

A sterile glass beaker containing an excess volume of sterile calcium chloride (0.65 - 1.5% w/v) is placed on a magnetic stirring plate below the spray head assembly such that the bottom of the spray head assembly is 5 - 10 inches about the surface of the calcium chloride solution. A sterile magnetic stir

~ -OS-ϋ-iasT bar is placed in the calcium chloride. The magnetic stir plate is set at low speed.

The outflow tube of the peristaltic pump (e.g. Rainin "Rabbit") is connected to the liquid inlet of the spray head assembly, (e.g. Spraying Systems 1/8 JACN) . The inlet tube of the peristaltic pump is inserted into the sodium alginate/cell suspension. The air tube is connected to the spray assembly and the air flow meter adjusted to 0-10 SCFH. The pump is turned on and adjusted so that the flow rate is 0-10 ml sodium alginate-cells/min. Droplets formed using this procedure fall into the solution of calcium chloride where sodium ions are replaced by the higher affinity calcium ions resulting in increased cross-linking of the alginate and formation of a stable calcium alginate gel containing entrapped hybridoma cells.

The procedure for washing alginate beads is as follows: After completion of the gelling reaction, the beads are allowed to settle out of suspension. The gelling solution is then removed by aspiration, and a volume of sterile physiological saline th ree times the volume of the settled beads added to the vessel. After the beads have resettled, the saline is aspirated and an additional three volumes of sterile saline added as above. This wash solution is aspirated, and two more washes are performed as above, except that sterile serum-free cell culture medium is used. Finally, the beads are resuspended in 5 - 10 volumes of culture medium containing all supplements (serum, L-glutamine, and antibiotics) and transferred to a culture vessel. The gel-entrapped hybridoma cells are incubated at 37C and allowed to grow to their optimum cell density. The culture supernatant is removed and replaced with an equal volume of fresh supplemented culture growth medium as needed. Continuous feed systems may automatically replenish the media on a continuous basis. The entrapped hybridoma cells produce and secrete monoclonal antibody into the surrounding culture media. At optimum cell densities (107 - 108 cells/ml of calcium alginate) , the hybridoma cells will produce antibody at the rate of lO-lOOjig/ml day or greater. The supernatant containing the monoclonal antibody may then be concentrated by conventional techniques to allow further purification of monoclonal antibody using techniques known by individuals skilled in the art.

The calcium alginate entrapped cells can be harvested and re-used by dilution of the calcium ions with chelating agents such as solutions of sodium citrate (10% w/v) ethylene diamine tetraacetic acid, (EDTA) , sodium salt of ethylene glycol-bis (- amino- ethyl ether N, N, N', N1 - tetra acetic acid) (EGTA) which sequester or chelate the calcium ions causing reformation of the liquid sodium alginate. The hybridoma cells can then be harvested from the sodium alginate.

From the forgoing it will be apparent that the process for proliferating cells in an entrapped environment and harvesting cell products theref rom can be practiced for a wide variety of cells and cell products without departing from the scope and spirit of the invention. The following examples should accordingly be construed in all respects as* illustrative and not in a limiting sense.

EXAMPL E I Human red blood cells were added to a 1.2% sodium alginate solution to give a final concentration of 2.5X105 cells per ml in 1.0% sodium alginate. The solution was then conveyed by a Rainin Rabbit peristaltic pump to a sprayer assembly. Compressed air was supplied at 20 PSI through air tubing to a Dwyer airflow regulator and thereafter through 0.22 Jim filter (M illipore Millex-GS) and then to the sprayer assembly. Droplets were formed at the sprayer assembly which contained human red blood cells. The droplets were deposited in a sterile beaker containing a 1.33% w/v calcium chloride solution from a height of 3 cm. The beaker was stirred with a magnetic mixer at low speed and the droplets were allowed to gel. The gelled droplets were allowed to remain in the gelling solution for up to 30 minutes. The gelling solution was thereafter washed and the gelled droplets were resuspended in medium containing various concentrations of py ruvate and/or adenine in HEPES buffer. Red blood cells have been preserved in this fashion without hemolysis for 60 days.


A hybridoma cell line was obtained from the

American Type Culture Collection (ATCC) . The cell line, designated as ATCC No. CRL-9017 (H25 B10) produces antibodies to Hepatitis B surface antigen (Ig

Gi Isotype) . It's culture medium was a Dulbecco's

Modif ied Eagles medium, 4.55 g/1 glucose; and Fetal Bovine serum, 10% or less. The gelling solution was prepared to have of final makeup of 1.3% CaCl2 -2H2θ, 0.5% glucose in 3mM HEPES pH 7.7. A first wash solution contained a 1:1 mixture of the gelling solution with 0.9% saline. The second wash solution contained a 1:1 mixture of the first wash solution with 0.9 saline. The sodium alginate-cell suspension was a mixture of 1.2% sodium alginate with one part of cell suspension to make a final concentration of 2.0 X 106 cells per ml of sodium alginate.

The hybridoma cells from ATCC No. CRL-9017 (H25 B10) were encapsulated as follows:

The sodium alginate-hybridoma suspension was conveyed through the silastic 1/16" I.D. tubing by the peristaltic pump which was set at 350 to give a rate of approximately 2.5 ml min to the sprayer. Air pressure was supplied to the sprayer assembly at 10-20 PSI. It was independently conveyed to the sprayer assembly by the 1/16" I.D. Silastic tubing through the Dwyer Gauge at 3 SCFH. From the air flow gauge the air passed through the Millex air filter to the spray - assembly. (NB. all fittings from the air filter to the spray assembly were autoclaved at 15 PSI for 15 minutes) . A beaker containing the gelling solution was placed directly below the spray assembly with a distance of about 3-4 cm between the spray orifice and. the surface of the solution. A magnetic stir bar was placed in the beaker and the gelling solution agitated at low speed. Droplets of the sodium alginate-hybridoma suspension formed at the oriface of the spray assembly and dropped into the gelling solution where they were allowed to remain for about 3 minutes after the spraying operation. The gelling solution was then aspirated and the gel beads were resuspended in the first wash solution and allowed to settle. After 5 minutes the solution was aspirated in the same fashion as before. The gel beads were resuspended in the second wash solution and again aspirated. Upon completion of the washing evolution, the gel beads were "placed in the above-described medium at a concentration of 20% gel beads.

The gel-entrapped hybridoma cells were incubated at 37^C and allowed to grow to optimum densities in approximtely 11 days. At 3 day intervals or when phenol red indicator changed to yellow, the vessel fluids were aspirated and f resh nutrient media added. Fetal Bovine serum supplement was reduced f rom 10% to 0% at the third change of medium. The sequestered cluster of hybridoma cells excreted monoclonal antibodies (IgG) into the medium. The medium containing these antibodies was removed for harvest. The fluids were concentrated with 50% ammonium sulfate and further purified through affinity columns. The gel-entrapped cells were placed in a sodium citrate solution which converted the calcium alginate gel to sodium alginate liquid, releasing the hybridoma cells which were gel entrapped in a repeating cycle or production.


A second hybridoma cell line was obtained f rom the ATCC with ATCC No. CRL-1644 (SJK-287-38) . This cell line produces antibodies reactive with DNA polymerase alpha. It's culture medium was Dulbecco's Modified Eagles medium (lOmM) 100 ml; glutamine lOOx, 1ml; non-essential amino acids, lOOx, 1ml; NCTC109, 10ml; Fetal bovine serum, 12ml; and 1ml of the solution prepared as follows: a) 1320 mg oxaloacetic acid b) 80 mg crystalline insulin (20 units/ ml; 25 units Img) c) stir (a) and (b) at 37C d) add 550 mg sodium py ruvate (50mM0 e) b ring to 100ml with distilled water and continue stirring until dissolved (Filter and Sterilize) .

These hybridomas were entrapped and propagated in gel beads as described in Example II.


A murine hybridoma cell line, designated as KTI-2A, produces antibodies to monoclonal antibodies. KTI-2A cell stocks were maintained in 500 ml spinner flasks in complete media (Iscove's media supplemented with 10% PB S, 6mM L-glutamine, pencillin (50U/ ml) and "streptomycin (50 meg/ ml) ) at 37C. Viabilities were > 90% . 2xl08 cells were prepared for gel encapsulation as follows by centrif ugation at 800 rpm for 5 minutes. The media was aspirated and the cell pellet was loosened by gently flicking the centrifuge tube. The cells were resuspended in 20 ml of 0.9% NaCl. Thereafter, 80 ml of 1.0% Na alginate

(Kelco- HV) was added" and the cells mixed to form an even suspension. The final sodium alginate concentration of the suspension was 0.8%

The cell suspension was then delivered to a conventional two-phase spray head using a peristaltic pump. Sterile air was also delivered to the spray

SUBSTITUTE SHEET head at 3.5 SCFH. The alginate/ cell droplets were propelled out of the spray head into a sterile beaker containing one liter of 1.2% CaCl2 to form gel beads. The gel beads were washed twice with 0.9% NaCl and once with complete medium. Thereafter the gel beads were transferred to a 500 ml spinner flask and brought to a 500 ml final volume with complete medium and cultured at 37θc Over a period of about two weeks, the cultures were fed as needed by replacing 50% of the spent- medium with fresh complete medium. The growth and viability of the gel entrapped hybridomas and antibody production are illustrated in Figure 2 as compared with unentrapped cells established in complete media at 2 x 106 cells/ ml. Entrapped cells were counted by dissolving a 0.5 ml aliquot of settled beads in 4.0 ml EDTA buffer (1% EDTA/0.5% NaCl, pH 7.1) . Cells were counted in a hemacytometer. Viabilities were determined by dye exclusion. Antibody concentration secreted into the medium was measured by ELISA.


Spontaneously transformed human B-cells, which secrete monoclonal IgM against human tumor cell antigen, designated KTI-7A, maintained in 850 cm2 roller bottles at 37C in a standard roller apparatus were entrapped in accordance with the procedure set forth in Example rv. The growth, viability and antibody secretion of entrapped v. non- entrapped B-cells over a two- week period is illustrated in Figure 3.

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Referenced by
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WO1987003616A1 *4 Dec 198618 Jun 1987Institut Francais De Recherche Scientifique Pour LMethod for cultivating microorganisms, particularly of the frankia group and preparation of bacterial inoculums
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International ClassificationC12R1/91, C12N11/12, C12N11/10, C12N5/02, C12P21/00, C12N11/04, C12P21/08
Cooperative ClassificationC12N11/04, C12N11/10
European ClassificationC12N11/10, C12N11/04
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