CA1280381C

CA1280381C - Entrapment of anchorage-dependent cells

Info

Publication number: CA1280381C
Application number: CA000512183A
Authority: CA
Inventors: Paul Vasington; Martin Sinacore; Robert Buehler
Original assignee: Schering Corp
Current assignee: Merck Sharp and Dohme LLC
Priority date: 1985-06-24
Filing date: 1986-06-23
Publication date: 1991-02-19
Anticipated expiration: 2008-02-19
Also published as: WO1987000197A1; EP0233899A1

Abstract

ABSTRACT

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 from a macro-environment, and harvesting the metabolic and/or other products or by-products.

Description

FI~L~-OF TH~ INVEN~IO~
The present invention relates to a process for entrapment, preservation and\or growth of anchorage-dependent cells and tissues in an artificial environment. More particularly, the present invention deals with methods and related products Eor entrapping anchorage-dependent cells and tissues in a permeable gel-like material, nurturlng 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 or growth of anchorage-dependent cells and tissues to high densities, increased yields of biologically produced products and many other benefits. Similarly, the present invention permits the entrapment and preservation of anchorage-dependent cells ~or long periods of time.

There are molecules of great investigative, clinical and perhaps commercial 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 artificial medium. The well-developed technology of industrial microbiology is adapted to the requirements of bacteria, yeasts and ,. ..
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molds. Each single cell, encased in a tough cell wall, is an independent metabolic factory with fairly simple nutritional requirements; for bacteria, glucose and some simple salts w~l often suffice. Microorganisms grow well floating free 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 fragile 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 defined. Rather than being a free-living organism, a mammalian cell is adapted to a specialized l~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 environment for each cell. Such a cell resists being separated from its tissue and grown in an artificial 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 ~n the laboratory. However, it has proved to be much more difficult to grow them efficiently on even a moderatPly larger scale.

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Techniques for moderate- and large-scale production of a~chorage-dependent animal cells have not changed significantly 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.
Current 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 facility to operate hundreds of these growth vessels for a single production run, even a simple manipulation such as medium supplementation requires hundreds of operations. ~ore 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, modified roller bottles, packed-bed propagators, artificial capillaries, microcarriers, and encapsulatian. Such techniques have been reviewed previously by Litwin, Proc. Biochem., 6:15 (1971), Maroudous, "New Techniques in Biophysics and Cell Biologyn, R.H. Pain and B.~.
Smith, Eds. ~Wiley, New York (1973)), and Levine et al.
~Cell Culture and its Applicationsn, R. Acton, Ed.
~Academic, New York ~lg77)).

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A more recen~ innovation in the propagation of anchorage-dependent cells is the microcarrier syste~.
The potentlally high surface-to~volume ratio (S/~ 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 environment and cell yield. A single reactor vessel also reduces laboratory space and manpower costs.
The microcarrier system is not without its problems, however. The potentially high S/V, and hence high cellular prod~ctivity, 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 manifested at low carrier concentrations (1 g A50/liter) as an initial loss of 50 to 75~ of the cell inoculum, and at higher carrier concentrations (> 2 g A50/liter) as greater degrees of cell loss and a general suppression of culture growth.
Various strategies have been employèd to alleviate the "toxic effectsn, including: pretreating the beads with serum or nitrocellulose, increasing cell inoculum, and adding spent culture medium or additives to the growth medium. It has been proposed that the observed A50 "toxicityn may be the result of the adsorption of certain critical nutrients by the beads. Others 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 _5_ ~ 3 ~
of the cells depending on the degree of attachment -strongly attached cells are often damaged or killed upon ~reatment with trypsin and sim;lar 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 leaching; (4) bridging between microcarriers resulting in mixing problems; and (5) recoverability o 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., M~t~ods in-Enzymolo~y, Vol.
44, Academic Press, New York, 1976; B.J. Abbott, ~n~.
Rpt~ Perm. Pr~c., 2:91 (1980); R.A. Messing, ~rih~J~
~erm. Proc" 4:105 (1980~; Shovers, e~ 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 protec~ed state within a membrane which is permeable to the plethora of nutrients and other materials required ~or normal metabolic functions.
One such technique is described in U.S. Patent No.
4,391,909, wherein tissue cells such as Islet of Langerhans cells are encapsulated within a spherical semipermeable membrane comprising a polysaccharide having acidic groups which have been cross~linked with acid reactive groups of a crosslinking polymer for permanence of the protective membrane. The semipermeable membrane has a selected limit of ~ 3 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 pro~ective 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 temporariIy gelled in a generally spherical shape by contact with the calcium solution. Thereafter, these "temporary capsules", are provided with permanent 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 constituen~s of the polysaccharides.
This technique has most recently been applied to a method of growing anchorage-dependent cells as disclosed in V.S. Patent No. 4,495,288, wherein the -7- ~ 3~
cell to be encapsulated is suspended in a medium containiny an anchoring subs~rate material and other high molecular weight components needed to maintain viability and to support mitosis prior ~o 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 satisfactory~
Moreover, cell densities thus far achievable within such membranes has been less than about 106 cells per milliliter of culture media. Both of these limitations affect the amount and recovery of useful and desirable cell products produced by the encapsulated material.
The ability to grow anchorage-dependent cells to hiyher cell densities within a protected ènvironment (capsule) would provide a means for achieving greater output of desirable cell products.
A further disadvantage of prior art methods of entrapping such cells is the inability to maintain cell viability at desirable higher cell densities. In addition, the restricted permeability of the capsular membrane prevents access of the encapsulated cells to high molecular weight inducer compounds. This restriction necessitates the release of the cells from capsules prior 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.

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.~LJM M ARY . OF TF ~ ~E~CI~
In accordance with the present invention, there is provided a novel approach to the entrapment, preservation and/or propa~ation 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 -vi~ro 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 andior proliferate anchorage-dependent cells entrapped therein. As noted above, it has been difficult to grow anchorage-dependent cells efficiently on even a moderately large scale wh~e 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 (Jarvis3~ it - .. - - . - . . ~

33~l g 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 interfere with the free diffusion into and out of the hydrogel beads. The added steps required to form ~he semipermeable membrane will have a negative effect on cell viabillties and make recovery of cells from capsules more difficult.
Also these "temporary capsules" must be nearly perfect spheres to insure formation of a non-leaking capsule.
The shape of the hydrogel bead in practicing ~he present invention is of less importance and has no direct bearing on the usefulness 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 thereinr simply by dissolution of the hydrogel, which leaves the cells intact, and free from any non-cellular materials. This cannot be easily achieved with microcarrier systems 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 -10~ 3 ~
cells prior to induction of product using high molecular weight inducers. Elimination of added steps will improve the subsequent cell viabllities and/or product formation.

BRIEF DE~cRI~-T-Io~ ~11 G~rlUlL~a Figure 1 illustrates one apparatus for entrapping anchorage-dependent cells.
Figure 2A depicts the growth and viability of entrapped murine epithelial cells designated Cl270 Figure 2B depicts secretion of hepatitis-B virus surface antigen from gel entrapped murine epithelial cells designated Cl?7.
Figure 3 depicts the growth and viability of entrapped murine fibroblast 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 muri~e 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.

~ETAII,ED DE~IPTION - QF _~Z~eIQ~
The present invention provides a novel approach for the entrapment, preservation and/or propagation of anchorage-dependent cells in---v~tr~ and harvesting products produced thereby. More spec~ically, it has now been discovered that anchorage-dependen~ 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 surrounding the entrapped cells; that such entrapped cells can be used to produce high levels of metabolic or other cellular products, such as hormones, vaccines, interferons7 and that, after a suitable period whereln 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 anchorag~-dependent cells are entrapped and propagated and their products harvested therefrom typically include the following steps:
A~ ~agQn~: (filter sterilized) 1. 1.0~ sodium alginate (Kelco-HV) in 0.
NaCl 2. 0.9~ NaC1

3. 1.2~ CaC12

4. Trypsin-EDTA solution (Flow Labs)

5. 1% EDTA/0.5% NaCl, pH 7.1

6. Complete culture media

7. Vitrogen-100 (Collagen Corp. Palo Alto, CA) B. ~ (standard sterile technique employed) 1. Anchorage-dependent cell stocks are maintained in 850 cm2 plastic disposable roller bottles or standard tissue culture flasks under conditions necessary to maximize cell viability (eg.
150 ml complete media per bot~le, 37C
incubation at a rotation rate of 0.25 rpm).
* Trade Mark . , :

C. ~xperimental Pr~t~ol (Standard sterile technique is employed throughout) l~ Cells are harvested from 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 37C and rotated at 0.25 rpm in a conventional roller apparatus.
2. After 10-20 minutes cells will begin ~o 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 ~he surface. The suspended cells are then counted in a hemocytometer. Typically a maximum of l-3 x 108 cells/roller bottle is obtained.
3. 1-5 x 108 cells (typically 2 x 108) are then centrifuged at 800 rpm for 5 minutes~
The media is aspirated off and the cell pellet is loosened by gently flickiny the centrif~ge tube. The cells are then resuspended in 20 ml of a collagen solution (Vitrogen-10 ~ which has been neutralized to pH SoO~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 ~, . . .

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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 perlod o~
time sufficient to permit cell attachment.
4, 80 ml of 1.0% Na algina~e 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.
50 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 SCFH.
The alginate/cell droplets are propelled out of the spray head into 0.5-l.OL 1.2~ CaC12 solution to form shape-retaining gel beads.
Flow conditions are adjusted so that the gel beads are left in CaCl~ for no more than 15 minutes.
6. The gel beads are then washed twice with 009%
NaCl solution and once with complete media.
7. Cultures are best established by resuspending the gel beads in complete culture media to 20-30% beads (v~v) and incubating a~ 37C
with mixing. Cultures are refed as neededO
Preservation of entrapped anchorage-dependent cells is accomplished by modifying the culture media, i.e. reducing the serum and/or glucose concentration to decelerate the growth o the entrapped cells.

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8. Cells are counted by washing a 0.5 ml aliquot of beads with 10 volumes of 0.9% NaCl and dissolving the bead~ in loS ml 1~ EDTA/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 37C
for 15-30 minutes with occasional shaking.
1.0 ml 0.4% trypan blue solution i5 added and the cells are counted in a hemocytometer.

9. Metabolic and other cell products may be harvested from the media where said products diffuse i~to the media. Entrapped cells may be released from the hydogel beads for final harvesting by adding 2-5 volumes of E~TA
buffer and incubating ~or 20 minutes at room temperature. Cell agregrates may be dispersed trypsinization.

Although the above-outlined steps represent the preferred mode ~or practlcing the present invention, it w~l be apparent to those skilled in the art that the above-described approach can vary in accoxdance with techniques known in the ar~.
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, poly HEMA, and others known in the art can be used to advantage in particular environmen~s.
Highly preferred are clari~ied 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%

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calcium, 0.01% magnesium, and 0.1~ potass~um~
Relco-Gel HV has the higher molecular weight, having a srookfield viscosity of about 400 ~1% solution) to about 250 (2% solution). Of these products, the Kelco~
Gel HV is highly preferred. Preferably, the hydrocolloid is further clarified by sequential f~tration through filters having pore sizes of 2.5 1.2 and 0.6 micxons, respectivelyl and s~erilized before use by passage through a ster~e filter having a pore slze of 0.45 microns or smaller.
The concentration of hydrocolloid in the mixture should range from about 0.5 to about 1.4%, preferably about 0.6 to 1.2%, most preferably about 0.7-0.9%.
This is considerably below percentages previously used~
and is believed to resul~ 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 Relco Gel HV~
and especially with Relco Gel LV.
The particular anchoring substrate used for propa~ation of anchorage-dependent cells w~l depend on the requirements of the cell being entrapped.
Exemplary water soluble anchoring substrates include collagen, a natural protein which is the chief constituent o~ connective tissue in animals, collagen plus fibronectin, histones, 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 pr~ferably 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 .1-1.0 mg/ml of alginate. Wa~er insoluble anchoring substrates may comprise up to 50% (V/~ of the final bead volume.
Preferably, 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, preferably generally spherically-shaped particles. Preferably, the gelled particles are mobile and thus can be arranged for convenient culturing, treatment and product extrac~ion. 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 resinsl 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 should not be exposed to extremes of temperature~
pH, or to toxic chemicals, or 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 , ,; .
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8. Various steps in treatment of the en~rapped 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 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 cells are preferably suspended in or otherwise contacted with a fluid containing oxygen, nutrients, vitamins, minerals, etc.t which can diffuse through the hydrophilic gel to the cells and thus maintain viability and growth. It may also be desirable to 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 culture 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 cource of sterile air, means for admixing the cells to be grown with the anchoring substrate/hydrophilic gel-forming material while such material is in liquid form, means for feeding the , . ~ :

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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 ~aratus 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 um (micron) or less, so as to sterilize the air used.
The ster~ized air then proceeds through a control valve 14, to a conventional two-phase spray head 15, where it mixes with the liquid cel~hydrocolloid mixture.
The liquid cel~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-OolOO m~) 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 tipso 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 th~ other alkaline earth metals ~except magnesium}, other divalent metals, and divalent organic cations, such as ethylene disamineO
Preferably, tank 17 and container 18 are both stirred ~: r ^, 1 l " f `~ ?

,' --19~ 38'~
during the process at slow speed, in order to keep the solids from settling out and to maintain constant concentratlon.
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 2mm 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 hydrocollo~d 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.
The spray head or nozzle utilized in connection with this invention need not be the modified hypodermic syrinyes 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 Spraying Systems, Inc., such as products sold under the designations 1/8 and JACN, lf8 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 t~,p orifice. Preferred inner diameters for the liquid spray tip include 0.006n, 0.010n, 0.016n, and range in size to a maximum of 0.100" with the smaller sizes preferred, to produce smaller droplets.

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The following examples are given to additionally ~llustrate embodiments of ~he present invention as it is presently preferred to practice. It w;ll be understood that these examples are illustrative, and that the lnvention is not to be considered as restricted thereto except as indicated in the appended claims.

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~ ntrapment---of - ~net~c~ nqineereds~-Mu~ine 1. Murine epithelial cells (clone C127 derivatives) were grown as monolayer cultures in 850 cm2 plastic roller bottles using media composed of Iscove's modification of DMEM supplemented with 10~
fetal bovine serum (FBS), 6mM L-glutamine, 50 units penicillin per ml and 50 micrograms streptomycin per ml ~complete media). 150 ml complete media per bottle was used and bottles were maintained at 37C a~ a rotation rate of 0.25 rpm.
2. Cells were harvested from roller bottles by trypsinization and counted.
3. 1.1 x 108 viable cells were centrifuged at 800 rpm for 5 min and the cell pellet was resuspended in 6.0 ml Vitrogen-10 ~ (pH6.0) collagen solution. The final concentration of collagen was 0.50 mg/ml sodium alginate.
4. Kelc ~ HV sodium alginate was added to a final concentration of 0.8% sodium alginate (i.e. 24 ml of 1%
HV sodium alginate). The final concen~ration of cells was 3.67 x 106 cells/ml alginate.
5. ~ydrogel~cell beads were delivered at 10 m~ min to a two-phase spray head (1650 head, 64SS air cap) with an air flow of 3.0 SCFH.
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6~ Hydrogel/cell beads were ~ ~ 0.50L 1.3~
CaC12, washed twice with normal saline and once wlth complete media.
7. Cultures were established at ratio of 20:80 tbeads: complete media~ in a T-flask and incubated at 37C in a humidified atmosphere containing 5% C02O
8. Cultures were fed as needed by replacing 50% of the spent culture fluid with fresh complete media. ~'h~
spent media was stored at -20C until assayed for antigen. Antigen was measured by radioimmunoassay~
9. Entrapped cells were counted by dissolving 1.0 ml of washed beads in 9 ml 1% EDTA/0.5% NaCl~
centr~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 37C, 0.5 ml 0.4~ trypan blue solution was added and the cells counted in a hemocytometer.
The growth, viability and antigen 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-lOO~as the anchoring substrate.

E-X*MP~E--II
~ ntr~pment ~f-Murine-Fib~ob~ast-~e~s 1. Murine fibroblast cells (clone SV-3T3; ATCC CCL
163.1) were grown as monolayer ~ultures in media composed of DMEM supplemented with 10% FBS, 50 units penicillin/ml and 50 micrograms streptomycin/ml (complete media).
2. Fibroblasts were harvested from bottles by trypsinization, counted and 6 x 107 viable cells were centrifuged.

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3. Cell pellet was resuspended in 6.0 ml histone II-A calf thymus (lmg/m~ (pH 7.0) and mixed w~th 24 ml 1% Kelco~HV sodium alginate. Final concentration of histone was 0.20 mg/ml alginate.
4~ All subsequent steps were as descr~bed in ~xample I, steps 5-10, with the exception of the RIA
quantitation of antigen in media.
The growth and viability of entrapped murine fibroblast cells (SV-3T3) is illustrated in Fig. 3.

~-XAMP~
~ ntr~ment~ um~-E-Ei~h~oi~-~ar~inoma-c~l~s 1. ~uman epitheloid carcinoma cells ~HeLa S3; ATCC
CCL 2.2) were grown as monolayer cultures in T-flasks in media composed of DMEM supplemented with 10~ FBS, 50 units pennicillin/ml and 50 micrograms streptomycin/ml.
2. Cells were harvested by trypSinizatiOn, counted and 1.2 x 108 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.

E-X-kMP~
~ntrapment-of Murine-M~mmary-~mo~ ell~m ~-lalnate-entrappe~-~h~h~i~L-~L~rQ~ao~icL~
1. Mouse mammary tumor cells were maintained in 850cm2 sterile disposable roller bo~tles in media composed of Iscove's modified DMEM (IM) plus 10% fetal bovine serum (FBS) r 6 mM L-glutamine, 50 units penicillin/ml and 50 mcg. streptomycin~ml (complete ~ v~

', , -23 ~ 3~
IM). Cell passages were carried out by incubation of monolayers with trypsin-EDTA solution.
2. Gelatin microcarriers (K.C. Biological, Lenexa, Ransas, catalogue ~MC-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, Ca2+, Mg2 free). The microcarriers were then washed twice in PBS
and mixed with 1 vol. PBS. Sterili~ation wa~ by autoclaving for 15-30 min. at 120C, 15 psi.
Microcarr~ers were stored at 4C ~n 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 ~orth in Example VI below. Mouse mammary tumor cells were trypsini~ed, 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. Af~er 15-18 hours at 37C the culture was divided into 2 equal aliquots and centrifuged. One pellet was resuspended in 1~5 ml complete media and used as unentrapped control culture. The other was entrapped as described hereinbelowO
3. Microcarriers were centrifuged at 80Drpm for 5 min. at room temperature and the supernatant was discarded. The pellet was resuspended in 1 3 volumes of sterile 0.8% sodium alqinate 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 8~
beads were-added to 3 volumes of complete IM in a spinner flask and incubated at 37C with gentle stirrlng. 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/D.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 37C 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 murine mammary tumor cells is illustrated in Fig. 5~

- ~*~MPL~-V
~ nt~pment-of-Chinese- ~mster-~ary~~eHO~ ~1-ls- i~
~ lginate-~nt~-a~pe-d ~e~ati~ ca~ç~rr-i-er~.
Genetically engineered CH0 cells were seeded onto 10ml of gelatin microcarriers (2x106 cells/ml gelatin) in a total culture volume of 125 ml. After 24 hours at 37C the culture was divided into control (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.

,, - ., _ . , . . :
. ' ', ` , . _ , : ' ~ ,, ' , ' ' 1 ~
.' " ;.. '.. `.. ' ' . ' ' . . .'' , ` ( 28~33~L
~-~kMP~
~ o~uc~on- ~f- ~-ros~link~- ~el~in- ~artic~e~
I. Reagents 1. gelatin-Type ~ porcine, 225 Bloom 2. glut~raldehyde-25% solution 3. distilled/deionized water 4. phosphate buffered saline, pH 7.4 (PBS Ca2~7 Mg2+-free).
II. Protocol 1. 100 gm gelatin was slowly added to 800 ml of rapidly mixing water preheated to 60 70C. 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 ~ 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 ~entrifuged 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 par~icles were ster~ized by autoclaving for 30 min. at 120C~

--26- 1Z~i~3 ~3f~
10. Crosslinked gelatin particles wQre stored in the dark at 4C and washed overnight with complete media just prior to use.

Claims

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

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) growing said ancnorage-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 from the group of collagen, fibronection, histones, poly-L-lysine or mixtures thereof is from 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-retaining 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 from 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 106 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 from the growth medium.

13. The process of claim 12, wherein the anchoring substrate is selected from the group of collagen, fibronectin, histones, poly-L-lysine, microcarriers or mixtures thereof.

140 The process of claim 13, wherein the microcarriers comprise particulate material selected from the group 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 from said growth medium.

17. The process of claim 15, including the additional step of releasing said cells from 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 preserving 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-dependent 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.