[go: up one dir, main page]

EP0233899A1 - Emprisonnement de cellules dependant de l'ancrage - Google Patents

Emprisonnement de cellules dependant de l'ancrage

Info

Publication number
EP0233899A1
EP0233899A1 EP19860904504 EP86904504A EP0233899A1 EP 0233899 A1 EP0233899 A1 EP 0233899A1 EP 19860904504 EP19860904504 EP 19860904504 EP 86904504 A EP86904504 A EP 86904504A EP 0233899 A1 EP0233899 A1 EP 0233899A1
Authority
EP
European Patent Office
Prior art keywords
cells
anchorage
droplets
dependent
dependent cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19860904504
Other languages
German (de)
English (en)
Inventor
Martin Sinacore
Paul Vasington
Robert Buehler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Karyon Technology Inc
Original Assignee
Karyon Technology Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Karyon Technology Inc filed Critical Karyon Technology Inc
Publication of EP0233899A1 publication Critical patent/EP0233899A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0012—Cell encapsulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2531/00—Microcarriers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/74—Alginate

Definitions

  • 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 itro 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.
  • the present invention permits the entrapment and preservation of anchorage-dependent cells for long periods of time.
  • 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.
  • 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.
  • anchorage-dependent 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.
  • 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.
  • 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 alleviate the "toxic effects", including: pretreating the beads with serum or nitrocellulose, increasing cell inocul um, and adding spent culture medium or additiv es to the growth medium.
  • 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 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 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.
  • 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
  • the medium is temporarily gelled in a generally spherical shape by contact with the calcium solution.
  • these "temporary capsules” are provided with permanent poly meric se m ipe meable membranes at their outer laye r, formed by perma nently Cro ss-linking or polymeriz ing the capsules wit h polymers containing reactive groups which can react with specif ic constituents of the poly sacchar ides.
  • 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.
  • th e 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.
  • SUMM ARY OR TH E 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.
  • Previous approaches to solving such problems have not been entirely successful, i.e.
  • 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.
  • 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.
  • 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.
  • the present invention a novel approach for the entrapment, preservation and/or propagation of anchorage-dependent cells in v itro 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,
  • 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 maximum of 1-3 x 10 8 cells/roller bottle is obtained.
  • 3. 1-5 x 10 8 cells 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.
  • 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.
  • crosslinked gelatin microcarriers 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.
  • 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% CaCl 2 solution to for m shape- retaining gel beads. Flow conditions are adjusted so that the gel beads are left in CaCl 2 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.
  • 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.
  • 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.
  • alginate which is a natural hydrocclloid derived from seaweed
  • 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% calcium, 0.01% magnesium
  • 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, hist ⁇ nes, 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
  • 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.
  • 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.
  • the gelled particles are mobile and thus can be arranged for convenient culturing, treatment and product extraction.
  • 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.
  • the entrapped cells shou ld not be exposed to extremes of temperature, pH, or to toxic chemicals, for amounts of time which would cause l oss 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.
  • 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.
  • an anchoring substrate in the media to optimize attachment and propagation of the entrapped anchorage-dependent cells.
  • Such substrates e.g. fibronectin
  • fibronectin are constituents of serum supplements normally used in cultur e fluids.
  • FIG. 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.
  • 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 um (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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Murine epithelial cells (clone C127 derivatives) were grown as monolayer cultures in 850 cm 2 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.
  • FBS fetal bovine serum
  • 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 106 cells/ml alginate.
  • 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% CaCl 2 , washed tw ice with normal saline and once with complete media.
  • 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.
  • entrapped murine epithelial cells C127
  • Figs. 2A and 2B 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.
  • 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) .
  • SV-3T3 entrapped mur ine fibroblast cells
  • 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.
  • 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.
  • HV sodium alginate
  • HeLa S3 human epitheloid carcinoma cells
  • EXAMPL E IV Entrapment of Murine Mammary Tumor cells in Aiqjnate- entrapped Gelatin Mi crocarr iers 1.
  • Mouse mammary tumor cells were maintained in 850cm 2 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 mcg. streptomycin/ml (complete IM) . Cell passages were carried out by incubation of monolayers with trypsin- EDTA solution.
  • 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.
  • PBS phosphate buffered saline
  • 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 6 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.
  • 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.
  • 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.
  • Fig. 5 The growth of alginate-entrapped gelatin microcarrier cultures of mur ine mammary tumor cells is illustrated in Fig. 5.
  • CHo cells were seeded onto 10ml of gelatin microcarriers (2x10 6 cells/ml gelatin) in a total culture volume of 125 ml . After 24 hours at 37°C the culture was divided into contr ol (unentrapped) and experimental cultures (entrapped) . Gelatin microcarriers were mixed with 3 volumes of 1% alginate and entrapped as described in Example IV.
  • the crosslinked gelatin was broken into large pieces, rinsed 3 times with 2 volumes of water and then mixed with 1 volume of water.
  • 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.
  • the gel particles were then resuspended in 2-3 volumes of a 0.1% gelatin solution and mixed overnight at room temp.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

Procédés et produits relatifs permettant l'emprisonnement de cellules et de tissus dépendant de l'ancrage dans un matériau perméable analogue à un gel, l'alimentation et la croissance de ces cellules dans le mini-environnement analogue à un gel tout en fournissant les éléments nutritifs nécessaires et d'autres matériaux à travers le gel perméable à partir d'un macro-environnement, et la collecte des produits métaboliques et/ou autres ou des produits dérivés.
EP19860904504 1985-06-24 1986-06-05 Emprisonnement de cellules dependant de l'ancrage Withdrawn EP0233899A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US74797785A 1985-06-24 1985-06-24
US747977 1985-06-24
US82360486A 1986-01-29 1986-01-29
US823604 1986-01-29

Publications (1)

Publication Number Publication Date
EP0233899A1 true EP0233899A1 (fr) 1987-09-02

Family

ID=27114846

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19860904504 Withdrawn EP0233899A1 (fr) 1985-06-24 1986-06-05 Emprisonnement de cellules dependant de l'ancrage

Country Status (3)

Country Link
EP (1) EP0233899A1 (fr)
CA (1) CA1280381C (fr)
WO (1) WO1987000197A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8705464D0 (en) * 1987-03-09 1987-04-15 Atomic Energy Authority Uk Composite material
EP0364606B1 (fr) * 1988-04-18 1994-03-16 Nitta Gelatin Inc. Procede pour la mise en culture de cellules animales a grande echelle, et procede pour la preparation du substrat de support a cet effet
DE3931433A1 (de) * 1989-09-21 1991-04-04 Hoechst Ag Verfahren zur kultivierung von zellen in mikrohohlkugeln
DE4038397A1 (de) * 1990-12-01 1992-06-04 Boehringer Ingelheim Kg Mikrocarrier fuer verankerungsbeduerftige zellen

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4409331A (en) * 1979-03-28 1983-10-11 Damon Corporation Preparation of substances with encapsulated cells
US4352883A (en) * 1979-03-28 1982-10-05 Damon Corporation Encapsulation of biological material
FR2470794A1 (fr) * 1979-12-05 1981-06-12 Pasteur Institut Nouvelles microparticules, leur preparation et leurs applications en biologie, notamment a la culture de cellules diploides humaines
US4407957A (en) * 1981-03-13 1983-10-04 Damon Corporation Reversible microencapsulation of a core material
US4495288A (en) * 1981-03-13 1985-01-22 Damon Biotech, Inc. Method of culturing anchorage dependent cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8700197A1 *

Also Published As

Publication number Publication date
CA1280381C (fr) 1991-02-19
WO1987000197A1 (fr) 1987-01-15

Similar Documents

Publication Publication Date Title
US4778749A (en) Tissue culture and production in permeable gels
CA2223417C (fr) Procede de fabrication de capsules d'un volume minimal contenant une substance biologique
Lim Microencapsulation of living cells and tissues—theory and practice
FI118853B (fi) Makrokapseloituja erittäviä soluja
Park et al. Microencapsulation of microbial cells
EP0741550B1 (fr) Revetements multicouche d'alginate pour tissus biologiques de transplantation
US5635609A (en) Particles prepared by transacylation reaction between an esterified polysaccharide and a polyamine, methods of preparation therefor and compositions containing same
US5429821A (en) Non-fibrogenic high mannuronate alginate coated transplants, processes for their manufacture, and methods for their use
US5620883A (en) Living cells microencapsulated in a polymeric membrane having two layers
NO163060B (no) Fremgangsmaate for fremstilling av substanser som dannes av levende celler som produserer interferoner eller antistoffer.
EP0127713A2 (fr) Micro-encapsulation de tissu vivant et de cellules
JPH0534946B2 (fr)
EP1011518A1 (fr) Transplant de tissu biologique recouvert d'un revetement d'alginate multicouche stabilise adapte a la transplantation et procede de preparation associe
EP0058689A4 (fr) Milieu de culture cellulaire, procédé de culture de cellules animales amelioré, méthode de production de microparticules et microparticules.
GB2094832A (en) Process for culturing anchorage dependent cells
Aydelotte et al. Culture of chondrocytes in alginate gel: variations in conditions of gelation influence the structure of the alginate gel, and the arrangement and morphology of proliferating chondrocytes
Tyler Microencapsulation of mammalian cells
US5387522A (en) Apparatus having a biphasic spray head for entrapping biological material in a hydrophilic gel
EP0185701A1 (fr) Culture et production de tissus dans des gels permeables
Kampf The use of polymers for coating of cells
CA1280381C (fr) Inclusion de cellules dependant d'un support
US8592203B2 (en) Selective siliceous bioreactor
Sakai et al. Proliferation and insulin secretion function of mouse insulinoma cells encapsulated in alginate/sol-gel synthesized aminopropyl-silicate/alginate microcapsule
Wang et al. Preparation and application of poly (vinylamine)/alginate microcapsules to culturing of a mouse erythroleukemia cell line
Sefton et al. Hydrophilic polyacrylates for the microencapsulation of fibroblasts or pancreatic islets

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19870527

RIN1 Information on inventor provided before grant (corrected)

Inventor name: BUEHLER, ROBERT

Inventor name: SINACORE, MARTIN

Inventor name: VASINGTON, PAUL