Active 3-D microscaffold system with fluid perfusion for culturing in vitro neuronal networks.

This work demonstrated the design, fabrication, packaging, and characterization of an active microscaffold system with fluid perfusion/nutrient delivery functionalities for culturing in vitro neuronal networks from dissociated hippocampal rat pup neurons. The active microscaffold consisted of an 8 x 8 array of hollow, microfabricated, SU-8 towers (1.0 mm or 1.5 mm in height), with integrated, horizontal, SU-8 cross-members that connect adjacent towers, thus forming a 3-D grid that is conducive to branching, growth, and increased network formation of dissociated hippocampal neurons. Each microtower in the microscaffold system contained a hollow channel and multiple fluid ports for media delivery and perfusion of nutrients to the in vitro neuronal network growing within the microscaffold system. Additionally, there were two exposed Au electrodes on the outer wall of each microtower at varying heights (with insulated leads running within the microtower walls), which will later allow for integration of electrical stimulation/recording functionalities into the active microscaffold system. However, characterization of the stimulation/recording electrodes was not included in the scope of this paper. Design, fabrication, fluid packaging, and characterization of the active microscaffold system were performed. Furthermore, use of the active microscaffold system was demonstrated by culturing primary hippocampal embryonic rat pup neurons, and characterizing cell viability within the microscaffold system.

SU-8 2000 rendered cytocompatible for neuronal bioMEMS applications.

Microfabrication advances have resulted in small, cheap, and precise devices for biological microelectromechanical systems (bioMEMS). SU-8/SU-8 2000 is an attractive material for these applications because of its high-aspect ratio fabrication capability, dielectric properties, and thermochemical stability. Despite these advantages, the potential toxicity of SU-8 2000 may limit its use in cell-based applications. We show that <10% of primary neurons survived when cultured adjacent to or on top of untreated SU-8 2000. We evaluated the efficacy of various detoxification and surface treatments for SU-8 2000 in neuronal cultures after 7-21 days in vitro. Viability was improved to 45.8% +/- 4.5% (mean +/- standard error of the mean) following 3-day heat treatment (150 degrees C) under vacuum, while UV exposure and CO2 supercritical extraction did not improve survival. Furthermore, parylene coating (25 microm), in combination with heat and sonication (in isopropanol) treatments effectively masked the SU-8 2000 and led to 86.4% +/- 1.9% viability. Glow discharge (oxygen plasma) treatment rendered the SU-8 2000 surface more hydrophilic and improved neuronal viability, possibly through improved cell adhesion. No organic leachants were detected by mass spectrometry before or after heat treatment or after sonication. However, XPS analysis revealed the presence of potentially neurotoxic elements, fluorine and antimony. Strategies to improve the cytocompatibility of SU-8 2000 with primary neurons will allow longer culture times and have applications for cell-based microfabrication.