Abstract:
A method of producing an improved cell growth surface and cell attachment surface. According to the present invention, a polymer article is molded at temperature in excess of 550° F. at the injection tip. After allowing the part to cool, a stream of plasma comprised of activated gaseous species generated by a microwave source is imparted on the article. This stream is directed at the surface of a polymer substrate in a controlled fashion such that the surface is imparted with attributes for cell adhesion superior to those of untreated polymer or polymer treated by other methods.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Application Ser. No. 60/817,814 filed on Jun. 30, 2006 and entitled “Method of Making Enhanced Cell Growth Surface” which is incorporated by reference herein. 
    
    
     BACKGROUND 
     The present invention relates generally to the field of cell growth laboratory ware and more specifically a product that facilitates cell growth. An apparatus and method for performing the surface treatment is also provided by the present invention. 
     The cultivation of living cells is a key component in, among other things, the drug discovery process. Many devices are sold for purposes of cell culture including roller bottles, flasks, dishes, multiwell plates, cell harvesting units, etc. Typically these items of laboratory ware are molded from polymers having a sufficient mechanical stability and strength to create the necessary substrate surface for cell attachment and growth. 
     Generally, cell growth containers or substrates need to be ‘surface treated’ after molding in order to make the surface hydrophilic and to enhance the likelihood for effective cell attachment. Surface treatment may take the form of a surface coating, but typically involves the use of directed energy at the substrate surface with the intention of generating chemical groups on the polymer surface. These chemical groups will have a general affinity for water or otherwise exhibit sufficient polarity to permit stable adsorption to another polar group. These functional groups lead to hydrophilicity and or an increase in surface oxygen and are properties recognized to enhance cell growth. Such chemical groups include groups such as amines, amides, carbonyls, caboxylates, esters, hydroxyls, sulfhydryls and the like. Examples of directed energy include atmospheric corona discharge, radio frequency (RF) vacuum plasma treatment, and DC glow discharge. These polymer surface treatment methods have displayed varying degrees of success and their effects tend to decay over time. 
     In the case of plasma treatment, plasmas are created when a sufficient amount of energy is added to gaseous atoms and/or molecules, causing ionization and subsequently generating free electrons, photons, free radicals, and ionic species. The excitation energy supplied to a gas to form a cold plasma can originate from electrical discharges, direct currents, low frequencies, radio frequencies, microwaves or other forms of electromagnetic radiation. Plasma treatments are common for surface modification in the microelectonic and semiconductor industries. As mentioned, atmospheric corona and RF plasma treatment are commonly used for polymeric surface activation for cell growth substrates as well as medical implants. 
     Current standard practices for growing adherent cells in cell culture involves the use of defined chemical media to which is added up to 10% volume bovine or other animal serum. The added serum provides additional nutrients and/or growth promoters. In addition, serum proteins promote cell adhesion by coating the treated plastic surface with a biolayer matrix to which cells can better adhere. The addition of serum is typically required to support the normal growth of the majority of cell lines. While advantageous for cell growth, serum can have adverse effects by introducing sources of infection or abnormally inducing expression of unwanted genes exposed to serum. 
     An advance over the standard practices details the use of microwave plasma surface treatment. In such a process, a stream of plasma is comprised of activated gaseous species generated by a microwave source. This stream is directed at the surface of a polymer substrate in a controlled fashion such that the surface is imparted with attributes for cell adhesion far superior to that of untreated polymer or polymer treated by other methods described above. This process is more fully described in U.S. Pat. No. 6,617,152 and 2003/0180903, the contents of which are incorporated herein by reference. Surfaces for cell culture which enhance cell attachment are desired. In addition, surfaces for cell culture which enhance cell attachment without the use of animal products such as serum are desired. 
     SUMMARY OF THE INVENTION 
     According to the present invention, a polymer part is molded at high temperatures relative to standard molding techniques. After cooling, the part is then subjected to a stream of plasma comprised of activated gaseous species generated by a microwave source. This stream is directed at the surface of a polymer substrate in a controlled fashion. The surfaces treated according to the present invention exhibit superior cell growth characteristics than those achieved by currently known methods. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic drawing of the microwave plasma treatment apparatus of the present invention. 
         FIG. 2  is a graphical representation of a comparative cell growth study performed with injection molded polystyrene microplates molded at a variety of temperature and with 2 different plasma gases. 
     
    
    
     DETAILED DESCRIPTION 
     Typically, injection molded polymer articles for use as disposable cell culture vessels are molded at temperatures of between 400° F. and 500° F. However, it has been discovered that maintaining all other standard molding conditions (pressure, fill time, cycle time, etc.) but raising the molding temperatures imparts characteristics in the article surface that, after proper post treatment processing, aids in the attachment and growth of cells. Although not intending to being bound by theories of operation, it is thought that molding at higher temperature creates a surface with greater texturing than those surfaces molded at relatively lower, industry standard temperatures. To that end, it is preferable that molding occur at temperatures in excess of 500° F., 550° F., and even in excess of 600° F. For purposes of this invention, ideal molding temperatures may vary depending on the type of polymer. For polystyrene, articles should be molded at temperatures of 550-650° F., 590-630° F., or 600-620° F. Cycle times for molding will typically run 12-17 seconds. Once removed from the mold and cooled, the articles are subjected to a microwave plasma stream. 
     With reference to  FIG. 1 , a basic construction of the microwave plasma stream apparatus for carrying out the method of the present invention is provided. A 2.45 GHz microwave generator  10  (MKS Astex, Wilmington, Mass.) serves as the energy source of this apparatus. The equipment preferably includes a generator, circulator, dummy load, tuner, and applicator. A gas line  12  connects to a gas source and delivers the process gas, which when sufficiently energized creates a continuous stream of activated or ionized gas. Suitable plasma gases include argon, nitrogen, oxygen, nitrous oxide, ammonia, carbon dioxide, helium, hydrogen, air and other gases known to those of skill in the art. A plasma chamber  14  serves as a manifold for the reaction between gas and microwave energy, and is in fluid communication with both the gas line  12 , via a valve  13 , as well as the microwave generator  10 . A conduit  16  connects the plasma chamber with a treatment chamber  18  through an aperture  20 . Within the first or outer treatment chamber  18 , a second or inner treatment chamber  22  is located. The inner chamber has a frusto-conical baffle section which serves to contain the plasma flow and direct it onto a part that is placed at its base. In this embodiment, the inner chamber shares a common base  25  with the outer chamber. The approximate 1-6 inch gap between the aperture and the neck of the second treatment chamber enable the plasma to flow out of the outer treatment chamber through a valved vacuum line  24 . A pneumatic elevating system  29  may be employed to move the base portion  25  away from the treatment chamber in order to remove treated parts and place new parts into the inner chamber in an automated fashion. The conduit  16  and outer treatment chamber may be made from conductive or nonconductive materials, especially quartz, aluminum or stainless steel. The inner treatment chamber may be made from a nonconductive material, and most preferably, quartz. 
     In operation, the apparatus of  FIG. 1  performs as follows: A molded polymer part to be treated is located within the inner chamber  22 . For purposes of illustration, a multiwell plate  26  has been placed on the base  25 , but the inner and outer chamber may be shaped, dimensioned and configured to accommodate any of a variety of polymer parts. A vacuum seal is created between the base  25  and the sidewalls  27  of the outer chamber. To enable continuous flow, vacuum pumping is maintained through the process. The valves  13 ,  23  are opened and the process gas is allowed to flow into the plasma chamber  14 , through the aperture  20  and into the dual chambered treatment area. The gas flows at a pressure preferably between 100 and 2,000 millitorr, and more preferably between 200 and 300 millitorr. The gas is preferably set to flow at a rate of 100 to 5,000 cc/min, and more preferably between 400 and 600 cc/min. While the process may run at any range of temperatures, it preferably runs between 40 and 150 degrees Fahrenheit and more preferably at room temperature, or approximately 72 degrees Fahrenheit. The microwave generator is engaged to create an output of between 300 and 10,000 watts, and preferably between 300 and 3,000 watts. The microwave energy entering the plasma chamber  14  interacts with the gas entering the plasma chamber resulting in activation of the gas thereby creating the resultant plasma. Due to the constant flow characteristics of the assembly, the plasma is directed through the conduit  16 , through the aperture  20 , and into the treatment chamber. The stream or jet created by the plasma flow through the conduit and aperture is directed into the outer treatment chamber  18 , subsequently into the inner treatment chamber  22 , and onto the polymer part  26  placed at the base  25  of the chamber. Flow out of both the inner chamber  22  and outer chamber  18  is assured due to the vacuum line  24 , which serves to evacuate the dual chambered treatment area. It should be noted that due to the inner treatment chamber  22 , the plasma stream is directed onto the part as opposed to directly toward the outlet valve  23 , thereby enabling the part  26  to have optimal contact with the stream. The inner treatment chamber  22  should be entirely enclosed and sealed from the outer chamber  18 , but for the opening at the neck. 
     The plasma is energized for between 1 second and 5 minutes and more preferably for between 5 and 20 seconds. Once treatment is complete, the microwave energy is ceased, valves are closed, an atmospheric vent valve  32  is opened to introduce nitrogen or dry air to the system and in order to return all the chambers to atmospheric pressure. After normalization of pressure, the part is removed by operating the pneumatic elevating system  29 . Optimally, a computer control system performs the steps outlined above in an automated fashion. After removal, the part is preferably given a standard sterilization treatment by exposure to gamma radiation. 
     The surface of the polymeric substrate to be treated can have any shape, for example it can be flat, curved or tubular. Preferably, it is a flat planar surface. For purposes of this invention, the polymeric substrate can be biodegradable or non-biodegradable. Preferably, to be useful in both in vivo and in vitro applications, the polymeric substrates of the present invention are non-toxic, biocompatible, processable, transparent for microscopic analysis, and mechanically stable. 
     A large variety of polymers may be used as substrates in the articles of the present invention. Examples of polymers useful in the present invention include polyacrylates, polymethylacrylates, polycarbonates, polystyrenes, polysulphones, polyhydroxy acids, polyanhydrides, polyorthoesters, polypropylenes, 
     polyphosphazenes, polyphosphates, polyesters, nylons or mixtures thereof. 
     Examples of substrates that can be treated by the method disclosed herein include but are not limited to: flasks, dishes, flat plates, well plates, bottles, containers, pipettes, tubes, medical devices, filter devices, membranes, slides, and medical implants. These items are typically formed by commonly practiced techniques such as injection molding, extrusion with end capping, blow molding, injection blow molding, etc. 
     Although the invention is targeted for cell adhesion, attachment, and growth, the resultant polymer substrate surface promotes adsorption of a number of biologically active molecules including but not limited to: peptides, proteins, carbohydrates, nucleic acid, lipids, polysaccarides, or combinations thereof, hormones, extracellular matrix molecules, cell adhesion molecules, natural polymers, enzymes, antibodies, antigens, polynuceotides, growth factors, synthetic polymers, polylysine, drugs and other molecules. 
     Any cell type known to one of skill in the art may be attached and grown on the treated substrates of the present invention. Examples of cell types which can be used include nerve cells, epithelial cells, mesenchymal stem cells, fibroblast cells, and other cell types. 
     While the mechanism for enhanced cell attachment to the substrate treated according to the present method is not fully understood, it is believed to stem from three general characteristics: surface morphology, chemical functionalities, and surface energy. 
     EXAMPLE 
       FIG. 2  is a graphical representation of a comparative cell growth study performed with injection molded 96-well polystyrene clear plates that have been molded at a variety of temperature conditions and subjected to two different types of microwave plasma treatments. A Cincinnati Milacron 300 ton injection molding machine was employed for making the plates that were later post treated with microwave plasma. The molding conditions were as follows: For “High Temperature” molding, temperatures at the injection tip were approximately 610° F. For “Cold Temperature” molding, temperature at the injection tip was approximately 570° F. For the “Cont” or control condition, a more standard molding temperature of 460° F. was employed. In “Type I” treatment, the plasma gas utilized was a nitrous oxide. The nitrous oxide generally imparts a negative charge to the treated surface. In “Type II” treatment, the plasma gas utilized was ammonia. Ammonia, when utilized as the plasma gas tends to impart a negative charge to the treated surface. Finally, all samples were compared to plates treated with known chemical cell attachment coating, poly-D lysine (PDL). Cells were seeded at 70,000 cells per well of the plate and tested in triplicate. Cell growth conditions were measured by optical density readings under 10% serum growth conditions for 24 hours. Optical density assay quantification was carried out by a standard calorimetric kit (Cell Titer 96-Aq., Promega Corporation, Madison, Wis.). The cell line used was Hek-293. Cells were seeded onto all surfaces at the same time, with the same initial number of cells, under the same conditions. Table 1 displays the data. 
                                             Molding Condition   Treatment Gas   Optical Density                       High   Nitrous Oxide   0.281               (Type I)               High   Nitrous Oxide   0.568           High   Nitrous Oxide   0.569           Low   Nitrous Oxide   0.250           Low   Nitrous Oxide   0.360           Low   Nitrous Oxide   0.421           Control   Nitrous Oxide   0.362           Control   Nitrous Oxide   0.277           Control   Nitrous Oxide   0.303           High   Ammonia   0.312               (Type II)               High   Ammonia   0.345           High   Ammonia   0.321           Low   Ammonia   0.349           Low   Ammonia   0.244           Low   Ammonia   0.269           Control   Ammonia   0.320           Control   Ammonia   0.380           Control   Ammonia   0.321           Poly-D Lysine   None   0.281           Poly-D Lysine   None   0.342                    
As demonstrated in the graph of  FIG. 2 , the microwave nitrous oxide plasma treatment molded at high temperatures significantly outperformed the plates molded at different conditions.
 
     From the foregoing description of the various preferred embodiments, it should be appreciated that the present invention may take many various forms and that the present invention is to be limited only by the following claims.