WO1983002954A1 - Supports multi-usages pour applications immunologiques et biologiques - Google Patents

Supports multi-usages pour applications immunologiques et biologiques Download PDF

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Publication number
WO1983002954A1
WO1983002954A1 PCT/US1983/000240 US8300240W WO8302954A1 WO 1983002954 A1 WO1983002954 A1 WO 1983002954A1 US 8300240 W US8300240 W US 8300240W WO 8302954 A1 WO8302954 A1 WO 8302954A1
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support material
material according
vitro
beads
solid
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PCT/US1983/000240
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English (en)
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Inc. Ventrex Laboratories
Bruce Jacobson
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Ventrex Lab Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • 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
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using 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
    • C12N2533/30—Synthetic polymers

Definitions

  • This invention relates to surface-derivatized solid polystyrenes including copolymers thereof with divinylbenzene, and their use as surfaces for cell culturing or immunological reactions.
  • the polystyrene is. a copolymer, in bead form, containing about 12% divinylbenzene. Both anchorage dependent and suspension cells may be cultured on these surfaces.
  • a small minority of mammalian cell types have been adapted for growth in suspension cultures. Examples include HeLa cells, BHK cells, mouse L cells and mouse myeloma cells. However, many other cell types have not been adapted for growth in suspension culture to date, and will grow only if they become attached to an appropriate surface. Such cell types are generally termed anchorage dependent and include endothelial cells of bovine pulmonary artery origin, mouse 3T3 fibroblasts, mouse bone marrow epithelial cells, mouse fibroblasts producing Murine leukemia virus, primary and secondary chick fibroblasts and normal human embryo lung fibroblast cells.
  • anchorage dependence in cells is often associated with a normal phenotype and a normal genotype
  • growth in suspension culture is often (though not always) correlated with the Invasive and metastatic properties of cancer cells, it is essential for large scale growth of many normal cells to develop efficient techniques of growing anchorage dependent mammalian cells in culture rather than attempting to isolate variants of anchorage dependent cells adaptable for suspension culture.
  • Microcarrier systems offer the advantage of having the highest growth surface to vessel volume ratio, and may advantageously be used in conjunction with interior surfaces of the vessels that contain them, as growth substrates and in solid state immunologlcal reactions, e.g., assays and immunological syntheses.
  • Dextrans of which Sephadex is an example, have since been used in a variety of forms as carriers, including beads with a positive charge (U.S. patents 4,189,534 and 4,293,654), positively- charged- dextran beads coated with polyanions (U.S. patent 4,036,693) or dextran beads crosslinked with proteins capable of forming a gel after crosslinking, such as gelatin or fibronectin (FR 2470794 ' , issued June 1981).
  • Dextran surfaces have a variety of properties that limit their usefulness in tissue culture. For example, they exhibit a large enough pore size to allow wasteful entry of essential cellular regulators, they tend to have substantial internal negative charges in their pores due to contaminating sulfated dextrans normally synthesized as a natural by ⁇ product in microbiological systems, and they are prone to swelling in the presence of aqueous solvents. Swelling makes it difficult to achieve chemical modification of substantially only the surface. Dextran surfaces cannot be used to maintain anchorage dependent cells at confluence, but instead tend toward wasteful sloughing off of these cells, especially when the surfaces are in bead form.
  • Polyacrylamide exhibits similar problems, e_. g_. , internal negative charges tend to build up in its pores from.hydrolyzed amides and positive charges build up in the pores from amination, It
  • Solid polystyrene beads with undefined surface properties have been employed as microcarriers [Sargent, G.F. , supra] .
  • the surface of a underivatized solid polystyrene bead is usually a low density mixture of biologically toxic compounds, such as ethers, epoxides, alcohols and carboxyl groups formed by glow discharge or ozonolysis.
  • Another support material disclosed by prior art as useful for the growth of cells in tissue culture is a commercial anion exchange resin made from styrene copolymerized with, e.g. , 2 to 4% of divinylbenzene, and then treated with a haloalkylating agent such as choromethyl methyl ether.
  • the haloalkylated product which is strongly basic, is then derivatized with an amine or a hydroxy compound to induce further, both surface and Internal, positive charges on the beads. See, for example, U.S. patents 3,887,430 and 4,266,032.
  • reaction of haloalkyl groups with amines or hydroxy groups is an equilibrium phenomenon which allows residual unreacted haloalkyl groups to ionize in aqueous media and poison any biological materials that may be attached to the
  • the parent application also teaches that when the beads are crosslinked with a high enough concentration of a crosslinker such as divinylbenzene to yield a molecular weight exclusion limit of about 350 daltons they have a pore size too small to permit entry of many important and expensive cellular compounds, e_. g_, vitamins, coenzymes, hormones such as insulin and growth factors, etc.
  • a crosslinker such as divinylbenzene
  • the substantial lack of charge within the pores aids in preventing the uptake of these compounds by preventing ionic binding of even those compounds of a size that can penetrate the pores. Since the uptake of these compounds is wasteful, a problem frequently encountered when carriers of the prior art are used for cell culture is eliminated.
  • the invention as herein taught encompasses the finding that solid polystyrene surfaces in any physical form, size or shape may be derivatized in the same way to produce surfaces having essentially the same characteristics which function with the same effectiveness in cell culturing and as solid surfaces for solid state immunological reactions.
  • solid polystyrenes useful in these techniques need not contain divinylbenzene or other crosslinkers in order to exhibit the desirable results contemplated by this invention in its broadest scope; however, the embodiment disclosed in the parent application involving the use of derivatized divinylbenzene crosslinked polystyrene in bead form as micro ⁇ carrier for cell cultures does possess the unique characteristic of being mechanically stable even ' during thin sectioning techniques required to process specimens for scanning electron or transmission electron microscopy, whereby these beads with, attached cells can conveniently be sectioned and
  • OMPI 1FO examined, per se, by these techniques, as disclosed in the parent application and illustrated in the .aforementioned Tissue & Cell article. This is of special importance because morphological study of the phenomenon of cell anchorage dependence is enabled thereby. Similarly intracellular viruses and other intracellular phenomena can be analyzed in situ in culture, without disruption from the microcarrier.
  • microcarriers in the present invention for preparation of specimens in scanning electron microscopy and In transmission electron microscopy provides a potentially highly reliable method of performing quality control, or otherwise monitoring the system, in the industrial use of the cell culturing methods of this invention.
  • the Invention in its broadest compass minimizes internal charge in the pores of the carrier surface and thereby enables greater production of cells from the same seed colony than other cell growth surfaces heretofore known.
  • a HeLa cell seed culture grown in a serum containing medium on the positively charged microcarriers of this invention yields up to four times the concentration of HeLa cell harvest relative to the same cell seed culture grown with a similar medium in suspension, and there is no lag period before growth commences, unlike suspension culture. This is surprising because suspension culture is widely believed to be the most efficient and most economical method of growing cells in culture.
  • the derivatized- carrier surfaces of this invention are hence reagents for substantially improving present methods in the cell culture art, from both the economic and technical point of view.
  • the present invention encompasses novel products, cells or immunogens covalently bonded to a uniform coating of a protein ionically bonded to a derivatized solid polystyrene surface.
  • the solid surface comprises a polystyrenedivinylbenzene crosslinked copolymer bead form which has been uniformly surface sulfonated to impart a negative charge and is then, either coated with serum proteins or a specific adhesion protein of compatible isoelectric focusing point or else is first reacted with a reagent which neutralizes the sulfonate and imparts a strongly - positive charge, ' e.
  • the new product is then formed by covalent bonding to a desired cell line or immunogen compatible with the serum or specific adhesion protein.
  • the positively charged plastic surface is coated with gelatin or another adhesion protein and cells are then firmly adhered and.spread with the purpose of cultivating the cells under conditions which promote production of desired antibody, enzyme, antigen, hormone, etc.
  • any solid polystyrene surface of any physical form, shape or size may be employed in the invention as broadly contemplated.
  • the solid may comprise any commercially available solid polystyrene surface and the polystyrene may be crosslinked, e.g., with divinylbenzene or another known crosslinking agent or else it simply may be of a high molecular weight. It may be in the form of a flat or curved sheet, or in a finely divided form or in any other shape, so long as it has a surface that can be derivatized and coated as herein described.
  • the preferred composition is a polystyrene crosslinked with about 12% divinylbenzene having a molecular exclusion limit of about 350 which is substantially free of internal charges. Beads with these characteristics have distinctively novel advantages In the art of culturing cells.
  • all of the cell products and processes of this invention are of significant economic advantage because they permit greater cell growth from a given seed culture with concomitant savings in expensive serum, nutrients, culture vessels, etc.
  • the immunogen products of this invention enable more efficient immunochemical reactions in a variety of specific applications.
  • BSA BSA ( ) ; or gelatin, (•) .
  • Figure 4 Light micrographs of cells attached to cell culture microcarriers. A, B and C; time course of cell attachment and spreading on gelatin coated beads. A is initial attachment; B is 15 to
  • F—igure 5 Effect o ⁇ f protein synthesis inhibitors on the incorporation of H-leucine into HeLa Cell proteins.
  • Cells were incubated in media containing various concentrations of cycloheximide (g ⁇ , puromycin ( ⁇ ) for 1 hr. or actinomycin-D (A) for 2 hrs. prior to the addition of radioactive leucine.
  • Figure 6 Effect of protein synthesis inhibitors on attachment of cells to gelatin coated beads.
  • concentration of inhibitors were actinomycin-D (G) 20 / «g/ml; puromycin 20 ___.g/ml ( ⁇ ) and cycloheximide C ⁇ ) 5 ⁇ g/ml. Cells were incubated in serum free
  • OMPI ° medium (SFM) containing the inhibitor for the indicated time at which point beads were added and the percent of cells attached determined 1 hr. later.
  • Figure 7 Effect of puromycin on morphology of cells as seen with phase contrast light microscopy.
  • A micrograph of untreated cell in SFM and B, cells treated for 2 hrs. with 20 a g/ml puromycin. Magnification 1000 X.
  • Figure 8 Effect of puromycin (20 ⁇ g/ml) on the attachment of HeLa cells to gelatin coated beads. Cells were incubated for 15 (A) or 45 ( ⁇ ) min in SFM containing puromycin before gelatin coated beads were added and the percent of cells attached was determined. Arrow indicates the time at which the beads were added. The percent of blebbed cells (_s) during the time exposure to the puromycin is given on the right.
  • Figure 9 Effect of cycloheximide (5 a g/ml) on the attachment of HeLa cells to gelatin coated beads in low (*) and high (s) shear conditions. Cells were incubated 1 hr. in the cycloheximide before beads were added. Washing the cells in cycloheximide free SFM before cell attachment assays had no discernible effects on the kinetics of attachment.
  • Figure 10 Effect of trypsiniza ion of HeLa cells on attachment to gelatin-coated beads.
  • Cells were treated with 5 ⁇ g/ml trypsin for 2 min. at which time trypsin inhibitor was added and the cells washed in SFM. Cells were incubated in SFM and at the times indicated an aliquot was. withdrawn and the percent cells attached within 1 hr. determined.
  • Control (1) trypsin-treated cells (#) ; trypsin- treated cells plus either actinomycin-D 20 ⁇ g/ml) or cycloheximide (5 -'g/ml) (E) .
  • This invention encompasses methods of culturing • anchorage- dependent cells on carriers, including but not limited to microcarriers.
  • the carriers of this invention can be tailor-made for a wide variety of requirements in the culture conditions of anchorage dependent cells.
  • the polystyrene surfaces used in this invention may be modified to have positive or negative surface charges of wide variation in density so as to enable the covalent attachment of various proteins associated with the phenomenon of anchorage dependence for specific cells.
  • a preferred type of microcarrier bead within this invention should have a molecular exclusion limit in the order of about 350, a feature which can reduce the waste of expensive cellular metabolites and regulators such as epidermal growth factor. It will be understood that the molecular exclusion limit may be substantially higher than 350 daltons, provided that the internal charge is small enough to substantially prevent internal ionic binding by metabolites and regulators. However, molecular exclusion limits substantially greater than 350 will allow some metabolites and regulators to enter the internal network of the bead, an undesired phenomenon.
  • carriers means solid surfaces comprising polystyrene, including polystyrene copolymers
  • microcarriers means small discrete particles or beads comprising such polystyrene. It is within the scope of the invention to utilize any commercially available solid polystyrene, Including any available copolymer, for the sulfonation step. It is also within the scope of the invention to obtain commercially a negatively charged, sulfonated solid polystyrene, Including a polystyrene containing a comonomer and then treat it further as herein described.
  • a suitable composition for example, is a solid polystyrene cross-linked with divinyl ⁇ benzene in a concentration preferably high enough to lower the molecular exclusion limit of the pores of the solid surface to about 350 daltons, e_. g_. , 12% divinylbenzene.
  • Polystyrene beads cross-linked with 12% divinylbenzene are available from commercial sources (Bio Beads-SX 12, BioRad Laboratories, Richmond, California). The presence of cross-linker is not necessary, however, and particularly not If the polystyrene is of a density and molecular weight such as to present a low porosity surface.
  • molecular weight exclusion limit or “molecular exclusion limit” or “limit” as used herein each mean the approximate molecular weight cut off below which molecules will freely pass through the cross-linked network of the carrier surface into the pores thereof without detectable retardation in flow rate.
  • the molecular weight exclusion limit can be measured by any group of compounds having molecular weights near the suspected value of the limit, e_. g_. , nucleosides or nucleotides for a molecular exclusion limit of about 350. De ⁇ termination of the limit is a standard practice. See, for example, Jones, G.D., "Chemical Alteration of Styrene Polymers". In: R.H. Bundy and R.F. Boyer (Eds. ) Styrene: Its polymers, co-polymers and derivatives, Hafner Publishing Co. , Conn. 1972, pp. 674-691.
  • the solid carriers are first modified to have a negatively charged coating to adapt to the requirements in culture of some kinds of cells.
  • Some anchorage- dependent cells can grow effectively in the presence of serum on negatively charged beads, for example, endothelial cells derived from bovine pulmonary artery.
  • the procedures for making the surface of the polystyrene beads negatively charged are based upon standard techniques used to generate strongly acidic polystyrene cation exchange resins such as the Dowex-50 series. To make these resins, a solid polystyrene is completely sulfonated after a few hours in concentrated sulfuric acid at about 140°C. [Jones, G.D. , 1972, supra] . If the temperature of exposure to the sulfuric acid is controlled, most of the sulfonation occurs at the surface. The density of surface sulfonation is also controlled by the time of exposure. The total number of sulfonate groups are determined by titration with base as well, as ionic adsorption of the positively charged dye methylene blue.
  • Methylene blue is able to penetrate surface pores having a molecular exclusion limit of about 350 daltons.
  • the number of surface sulfonate groups is determined by ionic adsorption of a dye that is too large to penetrate the pores.
  • Alcian blue is used to measure surface
  • C-.-H sulfonate groups when the molecular exclusion limit is less than about 1,000 daltons.
  • Dye adsorption isotherms are conducted according to known techniques [Jacobson, B.S. et al, Biochem. Biophys Acta 506, 81 (1978)]. It has been determined that Incubation at about 60°C. for between about 1.5 and about 2 hours is preferred for the preparation of sulfonated solid polystyrene containing about 12% divinylbenzene, In bead form. These conditions result in a surface charge density of about 1 microequivalent per gram.
  • the total charge is about 2-3 microequivalents per gram, but when the same beads are completely sulfonated under other conditions the total charge is about 5,000 micro- equivalents per gram, a quantity over three orders of magnitude higher. Without sulfonation the total charge is about less than 0.01 microequivalents per gram not substantially different from the internal charge after surface sulfonation of about 1-2 microequivalents per gram. It will be understood that substantially all of the internal charge after sulfonation under the preferred conditions is immediately underneath the surface which is bound by Alcian blue.
  • Surface sulfonated polystyrene is capable of supporting growth of endothelial cells from pulmonary artery in the presence of serum containing medium. It is contem ⁇ plated that the conditions for sulfonation can be varied to fit the substratum requirements of a particular cell type according to procedures well known in the art. For example, incubation at 60°C. for shorter lengths of time will yield a ' solid surface with lower surface charge density.
  • - ⁇ _TE_E_ sulfonated carrier surfaces can be modified to have a positively charged coating to adapt to the requirements in culture of some kinds of cells.
  • Some anchorage .dependent cells can grow effectively on protein-coated positively charged beads, for example HEL 299 (normal human lung fibroblast) , JLS-V9 cells, and endotheli.al cells derived from bovine pulmonary artery. Some cells adapted for growth in suspension culture will also grow on protein coated positively charged beads, " e_. ' g_. ,
  • Carriers .with, positively charged surfaces are made by incubation of the sulfonated carrier with any polyalkylamine having primary amino groups, followed by coupling with any of the standard coupling agents, such as one of the carbodiimides or glutaraldehyde.
  • the preferred carbodiimide is l-ethyl-3- (3-dimeth laminopropyl) carbodiimide.
  • Polyalkylamines having secondary amino groups may also be used with glutaraldehyde.
  • the preferred polyalkylamine for both coupling agents is poly ⁇ ethylenimine (PEI) , a polycationic amino compound that is commercially available.
  • PEI poly ⁇ ethylenimine
  • the preferred reaction conditions include a temperature between about 0 and about 30°C. , preferably about 20°C. and concen ⁇ tration of between about 0.01 and about 0.15 grams/ml of PEI at a ratio of preferably about 0.3 of grams PEI/grams dry sulfonated beads.
  • the pH should be between about 7.5 and about 10.5, preferably between about 9.5 and about 10.5, and stirring of the reactants should proceed for about 1 hour.
  • the beads are then washed and dispersed in about neutral buffer at'a concentration of about 6 grams dry sulfonated beads per 100 ml of buffer. Then between about 5 ml and about 15 ml of 10% glutaraldehyde is added with rapid stirring. Stirring is continued for about 1 hour, and the beads are washed with about neutral buffer.
  • glutaraldehyde see, ' for example, Wasserman, B.P. et al, Biotechhol. ' Bioehg. 22, 271 (1980).
  • the beads are then used for coupling of adhesion proteins or they may first be treated with PEI and sodium borohydride to block any remaining free aldehyde groups by standard and well known methods.
  • the range of conditions for ionically binding PEI to sulfonated beads in preparation for glutaraldehyde coupling are followed, except that the PEI is pre-equilibrated to a pH of about 4.5, and the pH of the reaction mixture is between about 4.5 and about 5.5, preferably between about 4.5 and about 5.0.
  • the mixture After adding PEI to the beads, the mixture is agitated for about 1.4 hours, then between about 0.05 ml and about 0.15 ml of IM l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is added dropwise for each g. of PEI beads. After shaking for about 24 hours at room temperature, about 0.015 g. of EDC is added for each g. of PEI beads. The concentration of carbodiimide is followed colorimetrically [Jacobson, B.S. et al, Anal. Biochem. 106, 114.(1980)]. The mixture is then diluted with enough water to about double the volume, and the beads are then allowed to settle.
  • EDC IM l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • the solution on top is aspirated, then about 5 ml of 3M NH/C1 per g. of beads, is added with stirring to neutralize any uncrosslinked amino groups. " After about 5 minutes, the bead slurry is filtered, washed with water and methanol, then stored. The beads can then be used for coupling to adhesion proteins or for cell culture in the presence of serum, wherein serum proteins inherently coat the beads before cell attachment occurs.
  • the carriers having either a negative or a positive charge can be treated to covalently attach specific adhesion proteins depending on the isoelectric focusing point of the proteins, or to attach other substances of like function such as the unidentified serum proteins which have long been known to facilitate cell attachment in the presence of serum containing media.
  • Adhesion of cells is known to be a complex process especially for those cells that must be attached to a substratum in order to be able to grow, ⁇ _. _. , anchorage-dependent cells.
  • Proteins known to be involved in the process of cellular adhesion for various different types of cells include, but are not limited to, collagen, fibronectin,. laminin, chondronectin and the proteoglycans. For instance, hepatocytes have been reported to bind to a combination of fibronectin and laminin [Johansson, S. et al, supra] .
  • ⁇ OMPI thereof bind to a population of anchorage-dependent cells, one synthesizes carriers covalently bound to the selected protein by known chemical procedures adapted to the peculiar physical requirements of the specific adhesion protein.
  • the type of carrier used in the coupling depends upon the Isoelectric focusing point of the protein.
  • the protein to be covalently attached is first completely adsorbed from solution by ionic interaction and then covalently bound to either the sulfonated or the PEI treated carrier.
  • Gelatin has an isoelectric focusing point of about 4.7 and is adsorbed ionically to the sulfonated surfaces, but not to the PEI treated surfaces if the pH Is kept below 5.0.
  • the density of substitution of adhesion proteins on the carrier surface of the carrier can be varied in any of a number of ways. Changing the degree of sulfonation will result in corresponding changes in surface density of negative charges. A more preferable way is to dilute the adhesion protein with an "inert" protein.
  • essentially inert proteins include, but are not limited to, bovine serum albumin (BSA), rabbit serum albumin (RSA) and dephosphorylated milk casein.
  • derivatized and proteinized solid polystyrenes of this invention can advantageously be utilized in a variety of specific applications, including but not necessarily limited to the following:
  • Example 9a requires finely divided solid substratum and hence is limited to a bead type substratum.
  • Polystyrene beads cross-linked with divinylbenzene (Bio Beads-SX 12, BioRad Laboratories, Richmond, California) were washed twice in four volumes of methanol and air dried in a fume hood to eliminate detergent used in their synthesis.
  • a quantity of 60 g of beads were added to 160 ml reagent grade H 2 S0, (95-99°) in a 500 ml round bottom flask, and was heated to 60 C with a heating mantle. The temperature was kept at about 60°C and the beads stirred either with a magnetic stirrer or by swirling the contents at 4-7 minute intervals.' After two hours the suspension was carefully poured into a 2 liter flask containing about 1.5 liters of water.
  • the bead mixture was suction filtered through a Buchner funnel with Whatman #1 paper yielding the named product.
  • the product beads were washed three to four times with about 1.5 ml of water until the filtrate was neutral. The beads were then washed in either methanol or isopropanol and air dried.
  • Example 2 Synthesis of Positively Charged Carriers By Carbodiimide Coupling
  • a 500 ml Erlenmey ' er flask containing 60 g of dry sulfonated beads was added 200 ml of 30 mg/ml polyethylenimine (PEI) previously adjusted with concentrated HC1 to pH 4.5 to 5.0 with pH paper.
  • the PEI had an average molecular weight of about 70,000 and was purchased as a 33%. solution from Polyscience Inc. , Warrington, Pa. If wet sulfonated beads were used they were transferred as a slurry to the 500 ml Erlenmeyer flask and allowed to settle.
  • PEI polyethylenimine
  • the solution above the beads was aspirated and 50 ml of 150 mg/ml PEI-HC1, pH 4.4-5.0 was added. .
  • the bead-PEI mixture was agitated on a rotary shaker for 1.5 hours and then 6 ml of freshly prepared IM 1- ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC) was added drop-wise. After shaking for 24 hours at room temperature 1 g of EDC was added and the beads shaken for four hours. The concentration of carbodiimide was followed colorimetrically and found to be properly in excess. The mixture was then diluted to 500 ml with water and the beads allowed to settle for about 2 hours.
  • Example 3 To the entire sample product of Example 3, in the form of the phosphate-washed beads, was added 250 ml of 10 mg/ml PEI.
  • the PEI solution was made by adding 8.0 g of 33% PEI to 250 ml 20 mM NaP0 , pH 7.0 followed by addition 2.2 ml concentrated HC1 which brought the pH between 8.5 and 9.0 according to pH paper. While the beads were stirring 0.2 g of sodium borohydride was added. The beads were rapidly stirred with a rotary shaker for an hour and then overnight with just enough stirring to keep them suspended. The beads were washed once in 3 M NH,C1, four times in water and twice in methanol or isopropanol before air drying.
  • Proteins were coupled to either sulfonated or PEI coated beads.
  • the charge of the microcarrier used in the coupling method depended upon the isoelectric focusing point of the protein.
  • OMPI A quantity of 40 mg .of gelatin was adsorbed to 1 gm of the product of Example 1 in 10 ml of 2 mM pyridine HC1, pH 4.5. Excess unadsorbed protein was washed away with pyridine buffer. The bead suspension was then brought to 50 mM in EDC using a fresh. 1 M stock solution and agitated on a rotary shaker for 1 hour at 22°C. The beads were washed twice in 100 mM NaPO ,, pH 7.2, and stored in the same buffer with 0.02% NaN 3 .
  • a quantity of 10 g of BSA was adsorbed to 1 g of the product of Example 3 in 10 ml of 2 mM pyridine HC1, pH 5.5. Unadsorbed protein was washed away with pyridine buffer. The bead suspension was brought to 50 mM EDC using a fresh 1 M stock solution and agitated on a rotary shaker for one hour at 22 C. The beads were washed twice in 100 mM NaPO , pH 7.2, and stored in the same buffer with 0.02% NaN 3 .
  • Example 6 Covalent coupling of proteins with pi below 7.5
  • the pH of the mixture was between 9.5 and 10.5.
  • the beads were washed three times in 1.2 liters of water and then dispersed in 1.0 liter of 20 mM NaPO,, pH 7.0, yielding PEI-beads.
  • a 1 gm sample of PEI-beads was resuspended in 20 ml of 100 mM NaPO, pH 7.5, to which 5 ml of 10% glutaraldehyde was added while the beads were rapidly vortexed. After 30 minute incu ⁇ bation the beads were washed once in the pH 7.5 phosphate buffer. The beads were then incubated in 10 mM NaCNBH- in 0.2 M NaPO,, pH 7.5, for 16 hours at about 21°C. The beads were washed once in 0.2 M pH 7.5 phosphate buffer and once in the 20 mM sodium phosphate pH 7.0.
  • a quantity of 2 g of gelatin was added to the beads in 15 volumes of 50 M sodium phosphate, pH 7.5, containing 5 mM NaCNBH_, with rapid vortexing. The mixture was left on ice for two hours and then the beads were washed several times in the 60 mM phosphate, pH 7.5.
  • HeLa-S cells were grown in spinner flasks fitted with a paddle wheel rotated with just enough speed to keep the microcarriers suspended.
  • the culture medium was RPMI-1640 supplemented with 5% calf serum.
  • the flasks were aerated with 5% C0 2 and kept at 37 C.
  • the beads were sterilized by washing in 70%, isopropanol and rinsed several times in medium before use. Attached cells were determined after they were released from the microcarriers by trypsinization in 0.05% trypsin . at 37 C for 10 minutes.
  • Endothelial originally from the bovine pulmonary artery were seeded onto microcarriers either directly from fresh isilates or from monolayer cultures as follows : to 1 liter borosilicate siliconized roller bottles (Siliclad, Clay Adams) , 3 ml of microcarriers suspended in Hepes buffer (approximately 3 million microcarriers) were added to 28 ml medium (M199 -f 10% FBS + antibiotics) . Cells were scraped from a T 75 flask with a rubber policeman (approximately 9 x 10 cells) and were then aspirated in a 1 ml pipette and seeded into the roller bottle.
  • the bottle was purged with N 2 ,--capped and placed in the roller bottle incubator (Bellco) set at the highest speed.
  • the medium was changed every 2 days.
  • the beads were re- suspended in fresh roller bottles (usually split 1:2), and fresh beads and medium were then added to make up the original volumes. The cells were found to colonize the fresh beads until confluence was reached again.
  • Endothelial cells which, had grown to con luence on each bead were prepared for electron microscopy by the following methods.
  • a sample (0.5 - 1 ml) of endothelium-covered bead suspension was withdrawn from the roller bottle and was placed in a 3 ml borosilicate glass test tube. The beads were allowed to settle for 1 - 2 mins.
  • the growth medium was aspirated and replaced with fixative, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.5, containing 6% sucrose. After vortexing lightly to ensure complete mixing, the beads were allowed to settle again and the fixative was replaced. Fixation was allowed to continue from 1 hour to overnight at 4 C.
  • the cell covered beads were washed in 0.1 M cacodylate buffer at pH 7.4, containing 6% sucrose (the usual concentration, 12% sucrose, was not added because the beads will not settle) . Post-fixation was carried out in 1% OsO, in 0.1 sodium cacodylate, pH 7.4, for 1 hour.
  • the beads were dehydrated through an ethanol series and embedded in Spurr's low viscosity embedding medium overnight at 67 C.
  • the cells on beads were fixed in glutaraldehyde as described above but containing, in addition, 0.2 to 2% tannic
  • SEM scanning electron misroscopy
  • the samples were fixed for 30 to 60 min in 2% glutaraldehyde in medium minus serum followed by 5 min. incubation with 1% -OsO, , pH 7.0.
  • the samples were dehydrated in a graded series of isopropanol or a yl acetate and critical point dried in a Polaron Critical Point Dryer Model E3000.
  • the dried samples were attached to the SEM stubs with two-sided Scotch tape and coated with, a 30 nm layer of gold in a Polaron Specimen Coater, Model E5000.
  • the samples were viewed with a Jeol JSM-25S scanning electron microscope.
  • HeLa-S cells were grown in suspension culture at 37° in a humidified 5% C0 2 incubator in RPM1- 1640 medium (Gibco Laboratories) supplemented with 5% calf serum, antibiotics (60 ug/ml penicillin, 100 ug/ml streptomycin), and 25 mM NaHCO-. These cells had a generation time of 22 h. Cells for experiments were taken from mid to late log phase of growth and, unlike those in the preceding examples were serum free when attached to the microcarriers.
  • Microcarriers (BIo-Beads-SX-12, 200-400 mesh, Biorad Laboratories, Richmond, CA) were made negatively charged by sulfonation of polystyrene beads and positively charged by coupling polyethylenimine to the sulfonate groups. Procedures as described in the foregoing example were used. Gelatin-coated beads were made as follows: 8.0 ml of 10 mg/ml
  • OMP y gelatin were added to 10.0 ml of 4.0 mM pyridine- HC1, pH 4.5, containing 0.2 g of sulfonated beads and incubated for 5 minutes with intermittent vortexing to keep the beads suspended.
  • the beads were washed 3 times with 10 ml portions of the pyridine-HCl buffer, resuspended in 19 ml of the same and 1.0 ml of freshly prepared 1 M 1-ethyl- 3(3-dimethylaminopropyl) carbodiimide (EDC) was added.
  • EDC 1-ethyl- 3(3-dimethylaminopropyl) carbodiimide
  • SFM serum free medium
  • SFM containing reagents as specified in the experiments, to a concentration of 1.5-2.5 x 10 cells/ml.
  • the microcarriers were kept suspended and moving by mounting the culture tubes containing the cell-bead - mixture on a rotary shaker. For high shear conditions 275 RPM was used and 150 RPM for low shear. Below 150 RPM some beads settled to the bottom of the tube.
  • the attachment assay was conducted in a modified spinner flask. The modifi ⁇ cation was accomplished by attaching a plastic paddle to the magnetic impeller and was designed to keep the cells suspended. Experiments done in the spinner flask were considered to be analogous to high shear conditions on the rotary shaker.
  • OMF centration of 1.5 - 2.5 x 10 cells/ml The cells were incubated with various concentrations of cycloheximide or puromycin for 1 h or actinomycin-D for 2 h. • H-leucine (47 Ci-/mmole) was added to a final concentration of 2 uCi/ml and the incubation continued for another 1 h. The cells were pelleted by centrifugation at 3000 x g for 10 min at 4 and the supernatant discarded. The pellet was dispersed into 0.1 N KOH and after 20 min cold TCA was added . o a final concentration of 10% to precipitate the proteins. The precipitate was washed with cold 10% TCA by filtration onto glass fiber filters.
  • OMPI OMPI The information above indicated that cycloheximide inhibited protein synthesis and cell attachment at concentrations which did not appear to af ect the gross morphology of the cells as puromycin did. Therefore, cycloheximide was considered .useful for determining whether the synthesis of proteins was required for both attachment and spreading.
  • the experimental approach was to determine the extent of cycloheximide inhibition of cell attachment to gelatin coated beads in high versus low shear conditions. The rationale was as follows: If cycloheximide inhibited cell attachment by inhibit ⁇ ing the synthesis of a cell surface component that binds gelatin it would result in a greater degree of inhibition of attachment at high compared with low shear.

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Abstract

Des polystyrènes solides sont dérivés sur leur surface uniquement avec des groupes sulfonate chargés négativement ou par ces groupes suivis de polyalkilamines chargés positivement, et sont ensuite recouverts de protéines, par exemple, par des liaisons covalentes, ces protéines étant associées soit avec une dépendance d'ancrage cellulaire soit avec un ancrage immunogène, et sont utilisés en tant que support pour des cultures cellulaires ou comme substrats pour des réactions immunologiques à l'état solide. Dans un mode de réalisation, des perles de copolymères réticulées de polystyrène divinylbenzène sont traitées de manière à obtenir des micro-supports particulièrement indiqués pour une culture cellulaire.
PCT/US1983/000240 1982-02-23 1983-02-22 Supports multi-usages pour applications immunologiques et biologiques WO1983002954A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0172178A1 (fr) * 1984-02-06 1986-02-26 Surface Concepts Pty Ltd Procede de culture cellulaire.
EP0245986A2 (fr) * 1986-04-28 1987-11-19 Rohm And Haas Company Immobilisation de cellules non dépendantes d'ancrage
WO1988001279A1 (fr) * 1986-08-15 1988-02-25 Commonwealth Scientific And Industrial Research Or Promotion de l'adhesion et de la croissance de cellules sur un substrat
FR2621128A1 (fr) * 1987-09-30 1989-03-31 Sanofi Sa Trousse et methode de dosage immunometrique applicables a des cellules entieres
EP0312135A2 (fr) * 1987-09-29 1989-04-19 Findley Adhesives Inc. Matrice en phase solide enduite avec un polymère et son utilisation dans les essais immunologiques
EP0341498A1 (fr) * 1988-05-02 1989-11-15 E.I. Du Pont De Nemours And Company Adsorbant contenant une surface de polyéthylèneimine dérivé par un groupe hydrophobe, sa production et son application pour essais immunologiques et séparations
AU592670B2 (en) * 1986-08-15 1990-01-18 Commonwealth Scientific And Industrial Research Organisation Promoting cell adhesion and growth on a substrate
FR2650951A1 (fr) * 1989-08-17 1991-02-22 Grp Interet Public Thera Agents activateurs de la secretion de l'insuline support de culture cellulaires et bioimplants comprenant lesdits agents
WO1992006380A1 (fr) * 1990-10-01 1992-04-16 Pharmacia Biosensor Ab Amelioration relative au dosage faisant appel a une liaison en phase solide
US5134177A (en) * 1991-05-02 1992-07-28 University Of Southern California Conducting composite polymer beads and methods for preparation and use thereof
US5436161A (en) * 1988-11-10 1995-07-25 Pharmacia Biosensor Ab Matrix coating for sensing surfaces capable of selective biomolecular interactions, to be used in biosensor systems
NL1004538C2 (nl) * 1996-11-14 1998-05-25 Cordis Europ Matrixmateriaal met meerdere biologisch actieve stoffen.
US6461825B1 (en) 1987-09-30 2002-10-08 Sanofi (Societe Anonyme) Immunometric assay kit and method applicable to whole cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0030885A2 (fr) * 1979-12-05 1981-06-24 Institut Pasteur Nouvelles microparticules, leur préparation et leurs applications en biologie, notamment à la culture de cellules diploides

Patent Citations (1)

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EP0030885A2 (fr) * 1979-12-05 1981-06-24 Institut Pasteur Nouvelles microparticules, leur préparation et leurs applications en biologie, notamment à la culture de cellules diploides

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Title
CHEMICAL ABSTRACTS, Vol. 84, No. 20, issued 17 March 1976 (Columbus, Ohio, US) T. MATSUDA et al.: "Modification and Characterization of Polystyrene Surface used for Cell Culture", see page 57, Abstract No. 136782y, Polym. Sci. Technol., 1975, (Biomed. Appl. Polym.) 135-46 *
CHEMICAL ABSTRACTS, Vol. 86, No. 4, issued 24 January 1977 (Columbus, Ohio, US) R.E. BAREISS et al.: "Cation Exchange (resins) in Porous Glass Beads", see page 29, Abstract No. 17395f, Angew. Makromol. Chem. 1976, 55(1), 129-39 *
CHEMICAL ABSTRACTS, Vol. 88, No. 10, issued 6 March 1978 (Columbus, Ohio, US) M. HOOK et al.: "Cold-Insoluble Globulin Mediates the Adhesion of rat Liver Cells to Plastic Petridishes", see page 314, Abstract No. 72157w, Biochem. Biophys. Res. Comm. 79(3), 726-33 *
CHEMICAL ABSTRACTS, Vol. 90, No. 16, issued 16 April 1979 (Columbus, Ohio, US) see page 51, Abstract No. 122741w, JP, A, 78137889 (Mitsubishi Rayon Co. Ltd.) 1-12-1978 *
CHEMICAL ABSTRACTS, Vol. 91, No. 13, issued 24 September 1979 (Columbus, Ohio, US) R.C. HUGHES et al.: "Molecular Requirements for the Adhesion and Spreading of Hamster-Fibroblasts", see Abstract No. 105967c, Exp. Cell. Res., 1979, 121(2), 307-14 *
CHEMICAL ABSTRACTS, Vol. 94, No. 10, issued 9 March 1981 (Columbus, Ohio, US) see page 41, Abstract No. 66661p, JP, A, 50127437 (Tokuyama Soda Co., Ltd) 2-10-1980 *
Experientia, Vol. 36, No. 25, issued 15 May 1980 (Basel, CH) F. GRINNEL: "Fibroblast Cell Substratum Interactions: Role of Cold Insoluble Globulin (Plasma Fibronectin)", see pages 505-507 *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0172178A4 (fr) * 1984-02-06 1988-01-28 Surface Concepts Pty Ltd Procede de culture cellulaire.
EP0172178A1 (fr) * 1984-02-06 1986-02-26 Surface Concepts Pty Ltd Procede de culture cellulaire.
EP0245986A3 (en) * 1986-04-28 1988-08-24 Rohm And Haas Company Immobilization of nonanchorage-dependent cells
EP0245986A2 (fr) * 1986-04-28 1987-11-19 Rohm And Haas Company Immobilisation de cellules non dépendantes d'ancrage
AU592670B2 (en) * 1986-08-15 1990-01-18 Commonwealth Scientific And Industrial Research Organisation Promoting cell adhesion and growth on a substrate
WO1988001279A1 (fr) * 1986-08-15 1988-02-25 Commonwealth Scientific And Industrial Research Or Promotion de l'adhesion et de la croissance de cellules sur un substrat
EP0312135A2 (fr) * 1987-09-29 1989-04-19 Findley Adhesives Inc. Matrice en phase solide enduite avec un polymère et son utilisation dans les essais immunologiques
EP0312135A3 (fr) * 1987-09-29 1990-04-04 Findley Adhesives Inc. Matrice en phase solide enduite avec un polymère et son utilisation dans les essais immunologiques
FR2621128A1 (fr) * 1987-09-30 1989-03-31 Sanofi Sa Trousse et methode de dosage immunometrique applicables a des cellules entieres
US6461825B1 (en) 1987-09-30 2002-10-08 Sanofi (Societe Anonyme) Immunometric assay kit and method applicable to whole cells
EP0341498A1 (fr) * 1988-05-02 1989-11-15 E.I. Du Pont De Nemours And Company Adsorbant contenant une surface de polyéthylèneimine dérivé par un groupe hydrophobe, sa production et son application pour essais immunologiques et séparations
US4952519A (en) * 1988-05-02 1990-08-28 E. I. Du Pont De Nemours And Company Protein immobilization with poly(ethyleneimine) derivatized with a hydroprobic group
US5436161A (en) * 1988-11-10 1995-07-25 Pharmacia Biosensor Ab Matrix coating for sensing surfaces capable of selective biomolecular interactions, to be used in biosensor systems
FR2650951A1 (fr) * 1989-08-17 1991-02-22 Grp Interet Public Thera Agents activateurs de la secretion de l'insuline support de culture cellulaires et bioimplants comprenant lesdits agents
WO1992006380A1 (fr) * 1990-10-01 1992-04-16 Pharmacia Biosensor Ab Amelioration relative au dosage faisant appel a une liaison en phase solide
US5716854A (en) * 1990-10-01 1998-02-10 Pharmacia Biosensor Ab Solid phase binding assay
US5134177A (en) * 1991-05-02 1992-07-28 University Of Southern California Conducting composite polymer beads and methods for preparation and use thereof
NL1004538C2 (nl) * 1996-11-14 1998-05-25 Cordis Europ Matrixmateriaal met meerdere biologisch actieve stoffen.
EP0853947A1 (fr) * 1996-11-14 1998-07-22 Cordis Europa N.V. Matériel matriciel contenant plusieurs matériaux bio-actifs

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