Abstract:
A method is disclosed herein for treating a polymeric surface to define an improved cell culture surface. The method includes the steps of: coating the polymeric surface with a hydrogel; and attaching proteins to the hydrogel-coated surface. Advantageously, a method is provided which consistently produces improved cell culture surfaces that generally avoid bare spots and possible undesired protein absorption or cell differentiation.

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
       [0001]    This application claims priority to U.S. Provisional Patent Application No. 60/954,916, filed Aug. 9, 2007, the entire contents of which are hereby incorporated by reference. 
     
    
     FIELD OF THE INVENTION  
       [0002]    The present invention relates to methods of treating polymeric surfaces with hydrogel supported proteins, for improved cell culture, and surfaces prepared by the same. 
       BACKGROUND OF THE INVENTION  
       [0003]    Bare plastic surfaces, such as polystyrene surfaces, typically do not provide a sufficient surface for growth of cell cultures. The incorporation of proteins on plastic surfaces helps to enhance adhesion and growth of cell cultures. In the prior art, such protein incorporation has been performed using passive coating of proteins on the surfaces. Passive coating, however, is known to have several problems, including denaturing, due to the change from a solution environment to a surface confined environment, and desorbing of coated proteins from the surface during cell culture, leaving bare spots. Passive absorption typically requires higher protein concentration in solution, likely due to low percentage of bound protein that has correct confirmation or orientation for cell adhesion. Barre spots oil the surface may undesirably absorb proteins from the cell cultures. In addition, bare spots may result in the undesired direct attachment of the cells on the bare plastic surface, resulting in possible cell differentiation. 
       SUMMARY OF THE INVENTION  
       [0004]    A method is disclosed herein for treating a polymeric surface to define an improved cell culture surface. The method includes the steps of: coating the polymeric surface with a hydrogel; and attaching proteins to the hydrogel-coated surface. Advantageously, a method is provided which consistently produces improved cell culture surfaces that generally avoid bare spots and possible undesired protein absorption or cell differentiation. 
         [0005]    These and other features of the invention will be better understood through a study of the following detailed description and accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES  
         [0006]      FIG. 1  depicts an improved cell culture surface found in accordance with the subject invention. 
           [0007]      FIGS. 2-5  are flowcharts representing various aspects of the subject invention. 
           [0008]      FIG. 6  is a flowchart representing an exemplary method in accordance with the subject invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0009]    With reference to  FIG. 1 , a vessel  10  is shown, which initially includes an, untreated polymeric surface  12 , which may be treated with the method herein to define an improved cell culture surface  14 . 
         [0010]    The polymeric surface  12  may be defined on a vessel  10  of any known configuration, such as a test tube, vial, flask,etc. Preferably, the polymeric surface  12  is the surface of a multiwell plate. For illustrative purposes, the vessel  10  shown in  FIG. 1  is a multiwell plate. More preferably, the polyrmeric surface  12  is a surface of a well of a multiwell plate. It is further preferred that the multiwell plate conform to conventional multiwell plate standards (e.g., the Standards of the Society of Biomolecular Screening) so as to be usable in drug assay handling equipment (e.g., high throughput screening (HTS) equipment). The multiwell plate can be formed with any number or arrangement of wells (e.g., 96 (8×12) wells). 
         [0011]    The term “polymeric surface” as used herein refers to any suitable such polymeric surface known to those skilled in the art. Suitable examples of polymeric surfaces include those obtained from polymeric hydrocarbons. As used herein, the term “polymeric hydrocarbon” is intended to refer to those polymers and copolymers obtained from repeating monomer units which are composed of carbon and hydrogen. The polymeric hydrocarbons may be saturated or unsaturated, and substituted or unsubstituted. Substituents may include atoms other than hydrogen and carbon, as long as they are present in an amount that does not detract from the substantially hydrocarbon nature of the polymer. Such substituents include acetal, halo, hydroxy, cyano, alkoxy, amino, amid o carbamoyl, and carbamido groups. Typical examples of a polymeric hydrocarbon surface include those made from substituted and unsubstituted polyethylene, polypropylene, polystyrene, ABS, PVC, polytetrafluoroethylene, polyvinylidene, and mixtures thereof. In a preferred embodiment, the polymeric hydrocarbon surface is polystyrene. 
         [0012]    The term “polymeric surface” is also intended to include surfaces obtained from those polymers containing one or more heteroatoms such as oxygen, nitrogen, or sulfur, in addition to carbon and hydrogen. Typical examples of such polymeric surfaces include surfaces obtained from substituted and unsubstituted polyethers, polyesters, polyamides, polyamines, polyimines, polyurethanes, polyrureas, polyacetals, polycarbonates, polyacrylates, polysulfides, polysulfones, and polysulfides. Also contemplated as being within the scope of the present invention are surfaces obtained from, polymers with backbones composed significantly of heteroatoms, such as silicones. 
         [0013]    With reference to FIG.  2 ., a method  16  of the subject invention is depicted, which includes a step  18  of coating the polymeric surface  12  with a hydrogel, and a step  20  of attaching proteins to the coated surface. Any known technique can be used to conduct the step  18  of coating the polymeric surface  12  with a hydrogel. It is preferred that the hydrogel be coated so as to be immobilized on the polymeric surface  12 . Preferably, the coating step  18  incorporates several intermediate steps as discussed below. 
         [0014]      FIG. 3  depicts a preferred method of achieving the coating step  18  in the present invention. In a first step  22 , functional groups are deposited on the polymeric surface  12 . Thereafter, in a second step  24 , the functional groups are exposed to hydrogel, preferably if) solution form. The functional groups may be any functional group desired, and preferably the functional groups are amine groups. 
         [0015]    As will be recognized by those skilled in the art, the step  22  of depositing functional groups on the polymeric surface  12  may be achieved by several methods. By way of non-limiting examples, the step  22  may include plasma or corona discharge treatment, gamma irradiation, electron beam treatment, polymer absorption, evaporative deposition, plasma polymerization, self-assemble monolayer formation, or a combination thereof. Preferably, the depositing of the functional groups step  22  is performed by plasma treatment or corona discharge treatment. 
         [0016]    With reference to  FIG. 4 , in a preferred embodiment, the step  22  of depositing functional groups includes two separate steps. In a first step  26 , a charge is imparted to the polymeric surface  12 , to produce a charged polymeric surface. The charge may be any desired charge, although in a preferred embodiment, the charge is a negative charge. Once the polymeric surface  12  is charged, a second step  28  involves exposing the charged surface to a polymer to form a polymerized surface. In a preferred embodiment, the polymer is an amine-rich polymer. More preferably, the polymer is a positively charged poly-amine, such as polyethyleneimine or polylysine, and the polymer is exposed to the surface using a solvent, such as water. Similar means to treat polymeric surfaces are included in commonly-owned co-pending U.S. patent application, Ser. No. 11/496,933, the contents of which are hereby incorporated by reference. 
         [0017]    The step  22  of depositing functional groups is complete once the polymeric surface  12  is exposed to the polymer to form a polymerized surface. Preferably, the resulting polymerized surface has a high density of functional groups. As stated above, the next step is the step  24  of exposing the polymerized surface to a solution of hydrogel. It is preferred that the hydrogel is in water solution or a buffered solution where the pH and ionic strength are suitable for a chemical reaction between the hydrogel and the functional groups oil the polymerized surface. It is understood that any hydrogel may be used with the present invention. Suitable hydrogels include polyvinyl alcohol, sodium polyacrylate, acrylate polymers and copolymers with an abundance of hydrophilic groups, polysaccharides and polysaccharide derivatives, such as dextran, and hyaluronan. It is preferred the hydrogel be stable and non-cytotoxic. 
         [0018]    The step  24  of exposing hydrogel to the polymerized surface may be achieved by any known methods. Preferably, the step  24  results in the hydrogel being immobilized on the polymerized surface. Various techniques for immobilizing the hydrogel may be utilized. Preferably, to immobilize tie hydrogel, the hydrogel molecules are activated to produce reactive functional groups. Any functional groups may be obtained, which are reactive to the functional groups in the polymerized surface formed by the step  22 . Preferably, the hydrogel molecules are activated to produce amine reactive groups. More preferably, a polysaccharide hydrogel, such as dextran, is oxidized with periodate to convert natural occurring hydroxyl groups to aldehyde groups. Periodate may be removed from the hydrogel solution by dialysis or column chromatography. 
         [0019]    Once the hydrogel solution is prepared, the polymerized surface formed by the step  28  may be exposed to the reactively activated hydrogel as per step  24 . In a preferred embodiment, the polymerized surface is an amine functionalized surface, and the activated hydrogel solution an amine reactive. As a result of the exposure, the reactive functional groups of the hydrogel solution become immobilized on the polymerized surface. In a preferred embodiment, oxidized polysaccharide hydrogel is immobilized on an amine polymerized surface. The immobilization may be achieved through any known mechanism Preferably, the immobilization is performed through the Schiff base formation between aldehyde and amine groups. The Schiff base can be stabilized by a reducing agent such as sodium borohydride (NaBH 4 ). Sodium borohydride also reduces the remaining aldehyde groups on the hydrogel to alcohol groups. This reduction step can be delayed until after the protein attachment step (step  20 ), so that the remaining aldehyde groups on the hydrogel can be used to react with the amine groups on the proteins. 
         [0020]    Once the step  18  is completed, with the polymeric surface  12  being coated with a hydrogel, as set forth above, the step  20  may be conducted to attach proteins to the hydrogel-coated surface. It is preferred that the proteins be attached so as to be immobilized on the hydrogel-coated surface. Proteins may be attached by any known technique.  FIG. 5  depicts a preferred method of attaching proteins to the surface. Preferably, the proteins are attached in two steps. In a first step  30 , the hydrogel-coated surface is activated to produce protein reactive groups. Any protein reactive group may be used, such as amine reactive groups, thiol reactive groups, carboxy reactive groups and the like. In a preferred embodiment, the hydrogel is activated to produce amine reactive groups. More preferably, the hydrogel has been activated to contain amine reactive groups before being immobilized on the polymerized surface; therefore further activation of the hydrogel is unnecessary. In a further preferred embodiment, the hydrogel may be an oxidized polysaccharide hydrogel, which has already been immobilized on the amine polymerized surface. In this further preferred embodiment, the hydrogel is further oxidized by periodate to enrich the aldehyde groups on the hydrogel surface. 
         [0021]    In a second step  32 , after the hydrogel surface has been activated, the activated hydrogel surface is exposed to a solution of proteins or protein mixtures resulting in attachment of the proteins or protein mixtures. Exposure of the proteins or protein mixtures to the hydrogel forms the cell culture surface  14 . In a preferred embodiment, the proteins or protein mixtures have amine groups. The preferred proteins or protein mixtures have lysine and/or arginine residues on the surface, which provide the amine groups. The proteins or protein mixtures may be attached to the activated hydrogel surface through any known means. Preferably, the proteins or protein mixtures are covalently attached to the hydrogel surface. The covalent attachment may take place through the reaction of the amine-reactive groups on the hydrogel surface and the amine groups on the protein or protein mixture surface. In a more preferred embodiment, the proteins or protein mixtures are covalently attached to the oxidized polysaccharide hydrogel surface. Such attachment may take place through the Schiff base formation between the aldehyde group on the polysaccharide hydrogel surface and the amine groups on the protein surface. Optionally, as discussed above, an additional step may be added whereby the Schiff base is reduced, i.e., stabilized, with sodium borohydride, sodium cyano borohydride, or the like. The proteins or protein mixtures may include human fibronectin, human collagen, human vitronectin, human laminin, human entactin, and other extracellular matrix proteins and protein mixtures, and combinations thereof. 
         [0022]    With reference to  FIG. 6 , a preferred embodiment of the present invention is depicted. In this embodiment, the polymeric surface  12  of the vessel  10  is initially provided, the polymeric surface  12  being polystyrene. The step  18  of coating the polymeric surface  12  with hydrogel is initiated with the step  26 , whereby a negative charge is imparted to the polymeric surface  12  by oxygen plasma treatment methods to produce a negatively charged surface  40 . Thereafter, the step  28  is conducted by exposing the negatively charged surface  40  to a 1% solution of polyethyleneimine. Exposure to this solution forms a polymerized surface  42 , more particularly an amine functionalized surface. Next, the polymerized surface  42  is exposed to a 20 mg/mL solution of oxidized dextran (hydrogel), to form a hydrogel coated surface  44 , per the step  24 . With the hydrogel coated surface  18  prepared, the step  20  of attaching proteins is conducted. The step  20  includes exposing the hydrogel coated surface  44  to a 10 mg/mL solution of periodate (step  30 ). Exposure to periodate further enriches the aldehyde groups on the surface of the vessel  10 , thus activating the hydrogel coated surface  44 . Thereafter, the activated hydrogel coated surface is exposed to a solution of proteins or protein mixtures (step  32 ) to form the cell culture surface  14 . In a preferred embodiment, a protein mixture is used which contains 0.1 mg/mL of human fibronectin and 0.05 mg/mL human collagen. 
         [0023]    The subject invention may have applicability in various contexts. By way of non-limiting examples, the subject invention can be used to prepare polymeric surfaces to obtain the following advantages: preservation of proteins on the surface by reducing the surface absorption-induced denaturing of proteins; prevention or reduction in the amount of proteins detaching from the surface; minimizing the non-specific absorption of proteins present in the cell culture medium, which therefore minimizes the risk of losing important components in cell culture medium; minimizing the undesired attachment of cells on bare surfaces. In stem cell culture, such advantages have a very important effect, as they may keep the cells from differentiation. 
         [0024]    In a demonstration using the preferred embodiments of the present invention, mesenchymal stem cells were cultured in serum free media, on both a polystyrene surface prepared in accordance with the present invention (as described above in connection with  FIG. 6 ) and a surface formed by passively coating an oxygen plasma treated polystyrene surface with the same protein mixture described above (i.e., 0.1 mg/mL of human fibronectin and 0.05 mg/mL human collagen). It was found that the number of cultured cells was six times greater on the surface of the present invention as compared to the surface formed by passive coating. 
         [0025]    In another demonstration using the preferred embodiments of the present invention, it was found that human embryonic stem cells can be grown on the surface of the present invention in an undifferentiated state.