WO1983002954A1 - Multipurpose supports for immunological and biological use - Google Patents

Abstract

Solid polystyrenes are derivatized on their surfaces with negatively charged sulfonate groups only or by such groups followed by positively charged polyalkylamines, and then are coated with proteins, e.g., by covalent bonding, which proteins are associated either with cellular anchorage dependence or immunogen anchorage and used as carriers for cell cultures or as substrata for solid state immunological reactions. In one embodiment, polystyrenedivinylbenzene cross linked copolymer beads are so treated to make particularly desirable microcarriers for cell culture.

Description

TITLE OF THE INVENTION: MULTIPURPOSE SUPPORTS FOR IMMUNOLOGICAL AND BIOLOGICAL USE

This application is a continuation-in-part of U.S. application Serial No. 351,425 filed February 23, 1981.

INTRODUCTION This invention relates to surface-derivatized solid polystyrenes including copolymers thereof with divinylbenzene, and their use as surfaces for cell culturing or immunological reactions. In one form of the invention the polystyrene is. a copolymer, in bead form, containing about 12% divinylbenzene. Both anchorage dependent and suspension cells may be cultured on these surfaces.

BACKGROUND OF THE INVENTION The techniques of growing mammalian cells in large quantity and at high cell density are essential to investigative work in the field of cell biology, and to the large scale development and isolation of pharmaceuticals and other valuable products derived from mammalian cells and for numerous other purposes. A typical example is the industrial use of these techniques to isolate enough human virus to make vaccines.

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. Since anchorage dependence in cells is often associated with a normal phenotype and a normal genotype, whereas 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.

The requirements of growth on a surface or substratum limit culturing conditions to only a small surface area relative to the volume of the flasks often used for culturing and to the quantity of culture medium which is employed. Cell culture plants in industry utilize a large number of low yield batch reactors in the forms of dishes, prescription bottles, roller tubes or roller bottles. Alternative surfaces for culturing include plastic bags, stacked plates, spiral films, glassbead propagators, artificial capillaries, and microcarriers. 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.

The growth of mammalian cells on microcarriers was originally reported by A.L. van ezel (Nature, 216, 64 (1967)). DEAE-Sephadex beads were used as the microcarrier, beads that are now known to be

O too porous and also subject to undesirable shrinking and deformation during preparation of cell cultures for transmission electron microscopy [Sargent, G.F. et al J. Microscopy, 122, 209 (1981)]. 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). Similarly, QAE-Sephadex, CM-Sephadex and Dowex-8X beads have been reported [Horng et al, Biotechnol. Bioeng. 17, 713 (1975)]. 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

- \JRE

_ OMPI \ VIPO undergoes undue swelling in aqueous solution and exhibits prohibitively high density when the molecular exclusion limit of its pores is near 350. High density polyacrylamide in bead form requires so much stirring of the culture to keep the beads in suspension that attached cells slough off or the beads break. Other solid substances used as cell or immunological carriers include porous silica (U.S. patent 3,717,551), a substance with very high density, and polyacrylonitrile (U.S. patent 4,024,020). See also, inter alia, U.S. patents 4,016,110, 4,115,537, and 4,126,669.

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. However, It is well known that 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

-^UKE OMPI ^es¬

treated areas. Furthermore, when protein is coated onto the outside surface before cells are attached, the protein coat will not bond to unreacted haloalkyl groups and hence will not completely and uniformly cover the surface.

Many cells have been reported to attach and spread on nonbiological substrata. For example, fibroblasts attached and spread on sulfonated polystyrene dishes (Martin and Rubin, Experimental Cell Research 85_, 319-333, 1974) while BHK and liver cells found oxidized polystyrene a stickable substratum (Klemperer and Knox, Lab. Pract. 2_6, 179-80, 1977). The degree of fixed charge on the surface of culture dishes has been altered and its relationship to adhesion of cells evaluated (Sugimoto, Experimental Cell Research, 135, 139-45, 1978; Maroudas, J. Cell. Phys. 9_0, 511-520, 1977). Unfortunately, all of these studies were done in serum containing media and it Is doubtful that the cells came in contact with the defined substratum upon initial attachment. More likely the substratum was coated by serum proteins (Grinnell, J. Cell. Biol. 56_, 659-665, 1978). It has been shown that in the case of the negatively and positively charged polystyrene beads different sets of serum proteins adsorbed and shielded the underlying surface charge (Jacobson and Ryan, Tissue and Cell, 14, 69-83, 1982). HeLa cells readily attached and spread on the positive but not the negative beads when serum was present (Jacobson and Ryan, id_.). It is recognized that there are several factors in serum that encourage cell attachment (cf., Grinnell, J. Cell. Biol. 56_, 659-665, 1978), therefore, any conclusion regarding the attachment characteristics of nonbiological substrata in the

^~ presence of serum must be done with caution.

The observation that HeLa cells attached to positively charged beads was not unexpected. Similar surfaces bound cells so tenaciously that upon cell rupture a sheet of plasma membrane was left attached to the bead surface (Jacobson, Biochem. Biophys. Acta 471, 331-5, 1977; Cohen et al, J. Cell. Biol. 75, 119-134, 1977).

Notwithstanding the variety of carriers known in the prior art, new and better types of carriers for cell culture are badly needed because the particular requirements for the culture of certain cells are not satisfactorily met by known carriers, including microcarriers. It is well known that the vast majority of mammalian cells and cell types cannot be cultured on a long term basis with heretofore known carriers. Moreover, previously used carriers have not reliably permitted growth of cell cultures after the cultures became confluent.

In parent U.S. application 351,425, filed February 23, 1982 there Is disclosed a method of treating polystyrenedivinylbenzene beads so that substantially all of their surfaces were derivatized while avoiding the buildup of substantial internal charge in the resin pores. Also disclosed is the novel use of these derivatized polystyrene 12% divinylbenzene copolymer beads as microcarriers for cell cultures, a novel product comprising the derivatized copolymer beads attached to the cells, and novel"methods of cell culture using the copolymer beads derivatized to adapt to the requirements of different cell types. 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. In addition, 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.

Subsequent to the filing of the parent application, essentially the same information it contains was published by the present inventor and a collaborator in Tissue & Cell, 14(1), 69-83 (1982).

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. The 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.

Information about the use of solid polystyrene beads to prepare cell monolayers for transmission electron microscopy was published about May of 1981 [Sargent, G.F., supra, 1981]. The solid polystyrene beads were reported to remain rigid during dehydration in the preparation of the specimens. At least inferentially, the article suggests that it is not possible to perform reliable morphological analysis of anchorage-dependence of cells nor of intracellular viruses, because the toxic nature of the surface of the solid poly¬ styrene bead will alter the morphology of cells attached. The present invention eliminates such toxic surfaces, allowing morphological analyses of high reliability. The use of the 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. Thus, as the parent application shows, 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. They are also an efficient means for expediting solid state immunological reactions involving the anchorage of antigens, antibodies, etc. to solid surfaces followed by their use as reagents, e_.g_. , in assay and synthesis systems where one reagent is desirably in solid form.

BRIEF DESCRIPTION OF THE INVENTION 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. In one special embodiment 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. g_, polyethylenimine, carbodiimide, and then with serum proteins or any compatible specific adhesion protein. In this embodiment the new product is then formed by covalent bonding to a desired cell line or immunogen compatible with the serum or specific adhesion protein.

In certain embodiments of this invention 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. Thus, 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.

When utilized in bead form, 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.

In general, 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. In addition, the immunogen products of this invention enable more efficient immunochemical reactions in a variety of specific applications.

DESCRIPTION OF THE DRAWINGS

The drawings are described as follows:

Figure 1. Kinetics of HeLa cell attachment and detachment in high shear conditions on polystyrene beads either positively charged (B) , negatively

(A) charged or coated with gelatin (0) .

Figure 2. Kinetics of HeLa cell attachment and detachment in high shear conditions on polystyrene beads coated with fibronectin, (A); la inin (SS) ,

BSA ( ) ; or gelatin, (•) .

Figure 3. Kinetics of HeLa cell attachment. in low shear conditions to polystyrene beads with a positive (£}) or negative (A) charge or coated with BSA (0) , gelatin (Δ) or laminin (f) .

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

30 min. later, and C is at 60 min. when full spreading is approached. D, cell pinching-off of a positively charged bead. Magnification 1000 X.

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 (gø , puromycin (Φ) 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. The concentration of inhibitors were actinomycin-D (G) 20_/«g/ml; puromycin 20 ___.g/ml (©) and cycloheximide CΔ) 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 (β) 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 μ 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) .

DETAILED DESCRIPTION OF THE INVENTION 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. For example, 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.

It is within the scope of this invention to anticipate the requirements in the culture condition for an anchorage dependent cell or suspension culture cell by ascertaining what adhesion proteins bind the cells by the methods of Kleinman, H.K. , J. Cell Biol. 83, 473 (1981) , and then to make a surface with a covalent coating of the protein or proteins that are found to bind the 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.

As used herein, the term "carriers" means solid surfaces comprising polystyrene, including polystyrene copolymers, and the term "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.

The terms "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.

According to the present invention, 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. Under these conditions, 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, whether or not in bead form, 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.

According to the present invention, the

-^_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_. ,

HeLa-S •

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. The density of surface positive charges is calculated from measure¬ ments based on proton titration according to well known methods.

For ionically binding PEI to sulfonated polystyrenedivinylbenzene beads in preparation for glutaraldehyde coupling, 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. For a discussion of coupling with 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. For ionically binding PEI to the polystyrene¬ divinylbenzene sulfonated beads in preparation for carbodiimide coupling, 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. 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. 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 conditions for imparting a positive charge to other solid polystyrene surfaces with PEI and glutaraldehyde or carbodiimide according to this invention are essentially the same as those for beads.

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] . Upon determining what adhesion protein(s) or combinations

■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. For example, 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. At pH values above 5.0 gelatin is adsorbed ionically to the PEI treated polystyrene surfaces but not to the sulfonated surfaces. In both cases, 1- ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) is used to covalently couple the protein.

In cases where the EDC coupling procedure results in the loss of function of the bound protein, another coupling procedure is available. PEI treated polystyrene surfaces are activated with glutaraldehyde and treated with cyanoborohydride to reduce the unstable amine-ldehyde addition product, i_. e_. , an imine. When an adhesion protein of the appropriate isoelectric focusing point is added, it ionically binds at the proper pH to the surface, and the thus formed addition product is then reduced with cyanoborohydride. This alternative procedure works for any protein with an isoelectric focusing point below about 7.5, about the maximum pH at which the cyanoborohydride can be used. At higher pH for coupling to the

OMPI PEI glutaraldehyde-activated surface, sodium borohydride is used instead.

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. Examples of essentially inert proteins include, but are not limited to, bovine serum albumin (BSA), rabbit serum albumin (RSA) and dephosphorylated milk casein. Thus, dilution of one of the proteoglycans with BSA will reduce the density of the proteoglycan on the surface.

The 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:

(1) As a support for culturing cell lines to produce larger cultures;

(2) As a support for specific cell lines capable of producing and expressing desired immunological products^' in vitro, e_. g_. antibodies or antigens, enzymes, etc.;

(3) As a support for immunogens such as antibodies or antigens to be utilized in solid immunoassays;

(4) As a support for solid state biochemical syntheses, e_. g_. , of polypeptides and the like.

The ensuing specific examples are particularly concerned with bead-form solid polystyrenes. It is to be understood that similar ani.onic and cationic derivatization and proteinization steps can readily be conducted by those of ordinary skill In the art on any solid polystyrene surface of any shape, size or form, and that the resulting prepared surface can then be utilized in the same manner as beads with one exception—the electron microscopy application, of Example 9a requires finely divided solid substratum and hence is limited to a bead type substratum.

Example 1 Synthesis of Negatively Charged Microcarriers

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 H₂S0, (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. For storage 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 To 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. 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. After aspirating the solution, 300 ml of 3M NH,C1 was added to the packed beads and stirred for five minutes. The bead slurry was vacuum filtered through Whatman No. 1 filter paper in a Buchner funnel. The beads were washed four times with one liter of water and twice with one liter of methanol. The treated beads may then be air dried or stored wet.

Example 3 Synthesis of Positively Charged Carriers Via Glutaraldehyde Coupling

A quantity of 60 g of dry sulfonated beads, a product of Example 1, was incubated with stirring in a solution of 50 g of 33% PEI plus 100 ml of water and 7 ml concentrated HC1. The pH of the mixture was maintained between 9.5 and 10.5. After one hour the beads were washed three times in 1.2 1 of water and then dispersed in 1 liter of 20 mM NaPO,, pH 7.0. While the beads were rapidly stirred 100 ml of 10% glutaraldehyde was added. Stirring was continued for another hour and the beads were then washed twice in phosphate buffer.

Example 4 Blockage of Free Aldehyde Groups on Positively Charged Microcarrier Beads

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.

Example 5

Covalent Coupling of Proteins to Microcarriers

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.

A. Covalent coupling of proteins with pi above about 5.0 "

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% NaN₃.

B. Covalen coupling of proteins with pi below about 5.0

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% NaN₃.

Example 6 Covalent coupling of proteins with pi below 7.5 A quantity of 60 g of sulfonated beads, the product of Example 1, were incubated with stirring in a solution of 50 g of 33% PEI plus 100 ml water and 7 ml concentrated HC1. The pH of the mixture was between 9.5 and 10.5. After one hour 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.

Example 7

Cell Culture of HeLa-S3 Cells

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% C0₂ 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.

Example 8

Cell Culture of Endothelial Cells

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 N₂,--capped and placed in the roller bottle incubator (Bellco) set at the highest speed. The medium was changed every 2 days. When the cultures were confluent, 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.

Example 9 Electron Microsopy

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. In some experiments the cells on beads were fixed in glutaraldehyde as described above but containing, in addition, 0.2 to 2% tannic

O acid. In all cases sections were cut on an LKB Ultrotome III with glass knives, picked up on unsupported 300 mesh grids and examined in a Philips EM 301 electron microscope.

For scanning electron misroscopy (SEM) 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.

Example 10

A. Cell Culture

HeLa-S cells were grown in suspension culture at 37° in a humidified 5% C0₂ 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.

B. Microcarrier Synthesis

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. This was incubated for two hours with constant mixing, washed once in 100 mM trietha- nolamine-HCl, pH 8.0 and once in 3 M NH,C1, three times in 0.1 M NaPO,, pH 7.2 and stored in the phosphate buffer with 0.02% a _o at 4°. Beads coated with BSA were made according to the same protocol as the gelatin beads, except that BSA was coated onto PEI beads, and the pyridine-HCl buffer was adjusted to pH 5.6. Laminin coated beads were made by ionic adsorption to PEI beads. An excess of laminin (Bethesda Research Laboratories, Bethesda, MD) , 1 mg, was mixed with 0.5 g of PEI-beads in 50 mM NaPO, , pH 7.2. After 15 min at 20° excess laminin was washed away with RPMI-1640 buffered with 25 mM HEPES to pH 7.3. The bound laminin fully coated the beads as indicated by shielding the underlying positive charge and from quantitative measurements of the amount that was adsorbed. Bound laminin was not desorbed from the beads by incubating in medium. Fibronectin beads were made by incubating 0.2 g of gelatin beads in 20 ml of calf serum (Gibco) for 1 hour at 37°. The beads were washed twice in RPMI medium and then used in experiments. Electrophoresis of protein eluted from these beads with SDS indicated that the bead surface had become saturated with protein a major fraction of which migrated as serum fibronectin. C. Attachment Assay

Cells were harvested by centrifugation and resuspended in serum free medium (SFM) or 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. Alternatively, 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.

Attachment assays were done at 37°, and the SFM was supplemented with 25 mM HEPES to maintain the pH at 7.3. The concentration of free cells in aliquots taken from the assay mixture at the appropri¬ ate times were determined using a hemocytometer, and from this the percent of total cells that had attached was calculated. There was no difference in the percent cells attached whether free cells or cells released from the beads after trypsinization were counted. D. Protein Synthesis and Inhibitor Studies

The effect of various protein synthesis inhibitors

3 on the incorporation of H-leucine into trichloroacetic acid (TCA) precipitable proteins was done according ' to the procedures of Sanduig et al. , J. Biol. Chem.

251, 3977-3984 (1976) ,- with minor modifications.

Cells were harvested and resuspended in Earle's minimal medium containing 5 ug/ml leucine to a con-

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. The radioactivity was determined by scintillation counting after the filters had dried. E.

were harvested and resuspended in 10 ml of 5 ug/ml trypsin (Sigma, from bovine pancreas, twice recrystallized, type III) in 9.6-mM EDTA, 137 mM NaCl, 2.7 mM KCI, 8 mM Na₂HP0,, pH 7.2 and incubated for 2 min at room temperature with gentle agitation to keep the cells suspended. The proteolysis was stopped by adding 20 ml of 12 ug/ml trypsin inhibitor in the above salt solution and incubating for 2 min. This amount of inhibitor was twice the amount found necessary to inhibit all of the trypsin activity. The cells were then pelleted and resuspended in 20 ml SFM or SFM containing a protein synthesis inhibitor. F. ^' Results

All attachment assays were done in serum-free media. This was done to insure that direct inter¬ action between cells and substratum was measured without the complication of interfering substances from the serum. Depending upon the type of micro- carrier- or culture dish, vastly different types of serum proteins have been shown to adsorb and in some cases completely shield the properties of the underlying surface.

1. Kinetics of Cell Attachment

Cells attached rapidly to gelatin coated beads when the two were incubated together in serum free medium under high shear conditions, and remained attached for the duration of the experiment (Fig. 1). Neither antibodies to laminin nor fibronectin interf red with this attachment and spreading suggesting that these proteins were not present on the surface of HeLa cells or if they were they were not involved in attachment to the gelatin. Cells incubated in high shear conditions with beads coated with nonbiological substances, such as polyethyleneimine (positive beads) or sulfonate groups (negative beads) , attached rapidly (75-80% after 15 minutes) but began to detach after 15-30 minutes (Fig. 1). Beads coated with proteins other than gelatin were also tested (Fig. 2) . As with gelatin, cells attached rapidly to BSA coated beads and remain attached. Attachment to laminin coated beads was slower than to the beads described above, with 60% attached by 30 minutes, after which detachment occurred. The rate of attachment to gelatin beads was inhibited when the beads were coated with fibronectin, only 10% attached after 15 minutes compared with 90% for gelatin. When the shear force of the assay conditions was decreased, by lowering the RPM of the rotary shaker upon which the assay tubes were mounted, detachment from positive, negative and laminin beads, did not occur, and the rate of attachment to laminin beads was increased (Fig. 3).

jllB

C TI Sjf-., V, i; c 2. Morphology of Cell Attachment In addition to the kinetics of attachment, the morphology of attached cells also depended on the type of bead surface. An examination with light microscopy was a convenient way to determine whether attached .cells were round, partially spread or fully spread. Full spreading was promoted by gelatin beads, but only to a very limited extent by others (less than 1Q%) . An example of cells con- ^■sidered to be on the way to full spreading is given in Figures 4A, B and C. Cells^' were round on all beads upon initial attachment and some were partially spread after about 15 min. During the remainder of the incubation, cells became fully spread on the gelatin but on other beads remained only partially spread, or else they began to pinch off. This was readily seen in the light microscope. An example of pinching-off cells is given in Figure 4D. Scanning electron microscopy and observations from fluorescent antibody staining of actin indicated that cells pinched off from positive, negative and laminin beads and in most cases it appeared that a portion of the cell was left behind.

3. Effects of Protein Synthesis Inhibition and Trypsin on Attachment to Gelatin Coated Beads

Cycloheximide and puromycin which inhibit translation of mRNA into proteins (Stryer, Bio¬ chemistry pp 641-667, Freeman & Co., 1981) were first evaluated for their effects on protein synthesis and then cell attachment and spreading. At concen¬ trations as low as 1 ug/ml of puromycin was required to achieve a similar effect. Maximal inhibition of

3 H-leucine incorporation into TCA precipitable material by actinomycin-D, an inhibitor of transcription of- NA occurred at 2.5 ug/ml and the inhibition could not' be increased by Incubating the cells in 20 ug/ml (Fig.5).

Treatment of cells with actinomycin-D did not influence their attachment and spreading on gelatin beads in high shear conditions (Fig. 6). On the other hand, treatment with optimum concentrations of cycloheximide decreased attachment in a time- dependent manner with 15% less cells attaching after 1 hour of treatment and 40-60% less after 2 hours (Fig. 6). Puromycin's effect on attachment was even more drastic than cycloheximide's with total inhibition resulting from two hours of treat¬ ment (Fig. 6).- However, caution must be used in interpreting these results as being caused by the Inhibition of protein synthesis since puromycin caused the cells to become extremely blebby (Fig. 7), whereas cycloheximide did not. Blebbyness was found to cause cells to dissociate from gelatin beads (Fig. 8). After being treated with puromycin for 15 minutes, cells attached rapidly to the gelatin beads and remained attached for about 45 minutes, at which time they blebbed and detached. Similarly, cells incubated for 45 minutes and then mixed with beads attached, but remained attached for only about 15 minutes before blebbing and falling off. Cells that were allowed to become blebby before being mixed with beads did not attach at all. Therefore, in the case of puromycin, inhibition of cell attachment could be due to bleb formation,and not a direct result of inhibition of synthesis of particular proteins. Fortunately, cycloheximide did not cause blebs but still inhibited cell attachment..

4. Cycloheximide Inhibition of Cell^" Attachment in High Versus^" Low Shear

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. This is because it would take more gelatin binding material to hold the cell to the gelatin coated beads under conditions where the forces would be in the direction of keeping the cell and bead mixture dispersed, i.e., high shear. Since the results fit the above (Fig. 9) in that cell attachment was inhibited at high but not low shear, it could be further rationalized that if the cells spread while in low shear, where the amount of gelatin binding material was known to be reduced by the cycloheximide, then protein synthesis was not required for cell spreading—at least within the time of the experiment. Such was found to be the case, spreading was not Impeded in low shear. It is also likely that the cycloheximide did not function by inhibiting the secretion of an attach¬ ment protein since there was no difference in the rate or extent of attachment when inhibitor treated cells were washed before addition to the gelatin coated beads. 5. Effect of Trypsinization on Cell Attachment Trypsin is commonly used to release cells from monolayer cultures. The protease causes, the cells to round up and dissociate from the culture dish (See e.g. Vogel, Experimental Cell Research^' 113, 345-354, 1978) . The attachment of HeLa cells to gelatin coated beads was inhibited by trypsinization (Fig. 10) . The trypsin concentration (5 ug/ml) and incubation time (2 min) were well below that routinely used to dissociate cells from culture dishes (See Tarone et al, J Cell Biol, 94, 179-186, 1982) Recovery from the trypsin treatment began within 2 hrs of incubation.in SFM, however, complete recovery did not occur and the degree of cell attachment leveled off at about 80 percent that of untreated cells. Both cycloheximide and actinomycin-D blocked the recovery. hile the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as would readily occur to those of ordinary skill in the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

^■^g 3 E

OMPI

Claims

WHAT IS CLAIMED IS

1. A solid support material for use in biologi¬ cal, biochemical and immunological processes compris¬ ing solid polystyrene or a solid copolymer of styrene which has been prepared by a series of steps which comprise,

(a) surface sulfonation to impart a negative ionic surface charge, followed by

(b) one or more steps by which a serum protein or specific adhesion protein Is uniformly coated thereon.

2. A support material according to claim 1, in which the surface sulfonation step is followed by a neutralization and protonation step with a polyamino compound, to produce a positive ionic surface charge before the performance of those steps which impart a uniform coating of a serum protein or specific adhesion protein thereon.

3. The support material of claim 1 in which the solid polystyrene or styrene copolymer has a molecular exclusion limit of about 350 daltons.

4. The support material of claim 2 in which the solid polystyrene or styrene copolymer has a molecular exclusion limit of about 350 daltons.

5. A support material according to either of claim 1 or claim 2 in which the solid polystyrene or copolymer thereof is crosslinked with about 12% divinylbenzene, is in finely divided particulate form, has substantially no internal charge as measured by the dyes alcian blue and methylene blue and has cells from an established in vitro cell culture attached thereto.

o p 6. A support material according to either of claim 1 or claim 2 in which the serum protein or specific adhesion protein is covalently bonded to the Ionically charged surface.

7. A support material according to either of claim 1 or claim 2 in which a specific adhesion protein is employed and is gelatin.

8. A support material according to either of claim 1 or claim 2 In which a specific adhesion protein is employed and is laminin. . A support material according to either of claim 1 or claim 2 in which a specific adhesion protein is employed and is fibronectin.

10. A method of culturing living cells in vitro on a support material accoring to claim 1.

11. A method of culturing living cells in vitro on a support material according to claim 2.

12. A method of culturing living cells in vitro on a support material according to claim 3.

13. A method of culturing living cells in vitro on a support material according to claim 4.

14. A method of culturing living cells i vitro on a support material according to claim 5.

15. A method of culturing living cells in vitro on a support material according to claim 6.

16. A method of culturing living cells in vitro on a support material according to claim 7.

17. A method of culturing living cells in vitro on a support material according to claim 8.

18. A method of culturing living cells in vitro on a support material according to claim .

OMF 19. A method of conducting an immunological reaction in vitro which comprises bonding an immuno¬ logical reactant to a solid support material accord¬ ing to claim 1 and proceeding with such reaction.

20. A method of conducting an immunological reaction in vitro which comprises bonding an immuno¬ logical reactant to a solid support material accord¬ ing to claim 2 and proceeding with such reaction.

21. A method of conducting an immunological reaction in vitro which comprises bonding an immuno¬ logical reactant to a solid support material accord¬ ing to claim 3 and proceeding with such reaction.

22. A method of conducting an immunological reaction in vitro which comprises bonding an immuno¬ logical reactant to a solid support material accord¬ ing to claim 4 and proceeding with such reaction.

23. A method of conducting an immunological reaction in vitro which comprises bonding an immuno¬ logical reactant to a solid support material accord¬ ing to claim 5 and proceeding with such reaction.

24. A method of conducting an immunological reaction in vitro which comprises bonding an immuno¬ logical reactant to a solid support material accord¬ ing to claim 6 and proceeding with such reaction.

25. A method of conducting an immunological reaction in vitro which comprises bonding an immuno¬ logical reactant to a solid support material accord¬ ing to claim 7 and proceeding with such reaction. 26. A method of conducting an immunological reaction in vitro which comprises bonding an immuno¬ logical reactant to a solid support material accord¬ ing to claim 8 and proceeding with such reaction.

27. A method of conducting an immunological reaction in vitro which comprises bonding an immuno¬ logical reactant to a solid support material accord¬ ing to claim 9 and proceeding with such reaction.

28. A method of conducting a solid phase bio¬ chemical synthesis procedure on a solid support material according to claim 1 which comprises bonding thereto a first biochemical reactant and then pro¬ ceeding with the remaining steps of the desired synthe¬ sis to prepare the desired end product.

29. A method of conducting a solid phase bio¬ chemical synthesis procedure on a solid support material according to claim 2 which comprises bonding thereto a first biochemical reactant and then proceeding with the remaining steps of the desired synthesis to pre¬ pare the desired end product.

30. A method of conducting a solid phase bio¬ chemical synthesis procedure on a solid support material according to claim 3 which comprises bonding thereto a first biochemical reactant and then proceed¬ ing with the remaining steps of the desired synthesis to prepare the desired end product.

31. A method of conducting a solid phase bio¬ chemical synthesis procedure on a solid support material according to claim 4 which comprises bonding thereto a first biochemical reactant and then proceed¬ ing with the remaining steps of the desired synthesis to prepare the desired end product.

oyp 32. A method of conducting a solid phase biochemical synthesis procedure on a solid support -material according to claim 5 which comprises bonding thereto a first biochemical reactant and then proceeding with the reamining steps of the desired synthesis to prepare the desired end product.

33. A method of conducting a solid phase biochemical synthesis procedure on a solid support material according to claim 6 which comprises bonding thereto a first biochemical reactant and then proceeding with the remaining steps of the desired synthesis to prepare the desired end product.

34. A method of conducting a solid phase biochemical synthesis procedure on a solid support material according to claim 7 which comprises bonding thereto a first biochemical reactant and then proceeding with the remaining steps of the desired synthesis to prepare the desired end product.

35. A method of conducting a solid phase biochemical synthesis procedure on a solid support material according to claim 8 which comprises bonding thereto a first biochemical reactant and then proceeding with the remaining steps of the desired synthesis to prepare the desired end product.

36. A method of conducting a solid phase biochemical synthesis procedure on a solid support material according to claim 9 which comprises bonding thereto a first biochemical reactant and then proceeding with the remaining steps of the desired synthesis to prepare the desired end product.