# Deposition of Cell Culture Coatings Using a Cold Plasma Deposition Method
###### Abstract  
Collagen coatings were applied onto polystyrene microplates using a cold atmospheric pressure plasma process. The coatings were compared to standard wet chemical collagen thin films using microscopy, surface energy, infra-red spectroscopy, electrophoresis, and cell culture techniques. Thin films were also deposited on gold electrodes using both coating methods and their structural and barrier properties probed using cyclic voltammetry. While the wet chemical technique produced a thicker deposit, both films appear equivalent in terms of coverage, porosity, structure, and chemistry. Significantly, the cold plasma method preserves both the primary and secondary structure of the protein and this results in high biocompatibility and cell activity that is at least equivalent to the standard wet chemical technique. The significance of these results is discussed in relation to the benefits of a single step plasma coating in comparison to the traditional multi-step aseptic coating technique. plasma polymerization; corona discharge; biomolecule deposition; collagen; protein voltammetry +
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Despite the ubiquitous nature of these tissue culture treatment surfaces, there are still significant differences observed between various commercial surface treatments [11; 12] and the simple carbonyl, amine, and carboxylic acid functionalities deposited on the surface do not fully replicate the complex interfaces found in vivo. These differences have driven advances to deposit larger, more complex molecules onto device surfaces. While higher energy plasma devices can be used to deposit thick layers of biocompatible ceramics such as hydroxyapatite [13], these techniques have limited applicability to temperature sensitive surfaces and cannot be used to apply proteins or polysaccharide layers. In order to overcome this limitation, a range of protein coatings and application approaches have been developed to further improve the biocompatibility and functionality of cell culture surfaces. These coatings are traditionally applied using well-established multistep protocols [14; 15]. The first step is often the creation of reactive sites on the substrate surface using a plasma treatment similar to those used in cell culture ware [16; 17; 18]. Aseptic wet chemical techniques are then used to apply the target biomolecule as a thin aqueous layer on the surface and the materials are then incubated for 1-24 h under laminar flow hoods. The excess liquid is then carefully extracted without scratching the surface and various washing or fixing steps then follow. In some instances, linker chemicals can be used to selectively tether biomolecules to the target surface [19]. These techniques allow for the attachment of collagen, fibronectin, laminin, and other materials to plastic, glass, or ceramic cell culture ware. Similar technology is also used to attach enzymes and antibodies to various surfaces for biosensor and diagnostic applications [20]. The overall multistep coating processes are complex, labor intensive, and require complex aseptic manufacturing lines. Recent developments in plasma chemistry have shown that nonthermal helium plasma sources operating at atmospheric pressure can be used to deposit large functional molecules on to various surfaces in a single step [21; 22; 23]. This opens the door to a range of surface modification options that are rapid, scalable, and economical while also retaining functional chemistry. BioDep is a recently developed single step, cold plasma process to deposit various biomolecule coatings onto surfaces [24; 25]. The technique uses a low energy plasma of inert gas to simultaneously activate both the target surface and a stream of aerosol particles that contain the biomolecule of interest as it passes through the plasma. As the activated aerosol droplets contact the target surface, the biomolecules contained within are deposited as a dry, coagulated coating.