Abstract
It has been demonstrated that neuronal cells cultured on traditional flat surfaces may exhibit exaggerated voltage gated calcium channel (VGCC) functionality. To gain a better understanding of this phenomenon, primary neuronal cells harvested from mice superior cervical ganglion (SCG) were cultured on two dimensional (2D) flat surfaces and in three dimensional (3D) synthetic poly-L-lactic acid (PLLA) and polystyrene (PS) polymer scaffolds. These 2D- and 3D-cultured cells were compared to cells in freshly dissected SCG tissues, with respect to intracellular calcium increase in response to high K+ depolarization. The calcium increases were identical for 3D-cultured and freshly dissected, but significantly higher for 2D-cultured cells. This finding established the physiological relevance of 3D-cultured cells. To shed light on the mechanism behind the exaggerated 2D-cultured cells’ functionality, transcriptase expression and related membrane protein distributions (caveolin-1) were obtained. Our results support the view that exaggerated VGCC functionality from 2D cultured SCG cells is possibly due to differences in membrane architecture, characterized by uniquely organized caveolar lipid rafts. The practical implication of use of 3D-cultured cells in preclinical drug discovery studies is that such platforms would be more effective in eliminating false positive hits and as such improve the overall yield from screening campaigns.
Highlights
A key goal in cell-based assay technology is to achieve cellular responses to external stimuli that are physiologically relevant to what happens in vivo as closely as possible
Porous polymer scaffolds with equivalent average pore sizes of 60–100 mm in diameter were fabricated. This pore size range was empirically found to be ideal for mouse superior cervical ganglion (SCG) cells, which are approximately 10 mm in diameter
As discussed in Cheng et al [23], we used a polymer to salt ratio of 1:20 to achieve the optimal light transmittance (80% in wet condition) while maintaining adequate mechanical strength, which is higher than the maximum possible force a typical fluid transfer workstation (e.g. FLEXstation, Molecular Devices, Sunnyvale, CA) could generate (0.11 mN)
Summary
A key goal in cell-based assay technology is to achieve cellular responses to external stimuli that are physiologically relevant to what happens in vivo as closely as possible. Many wholecell-based assays in use today rely on flat, two-dimensional (2D) glass or plastic substrates that may not produce results characteristic of in vivo conditions. Three dimensional (3D) substrates or scaffolds provide cells with in vivo-like topographical cues and enable cells to differentiate into specific phenotype and maintain specific functions that are usually impossible under 2D cell culture conditions [1,2,3]. 3D cell-based assay systems are desirable in preclinical drug discovery applications. Various approaches and materials have been studied for creating three-dimensionality. Among them are microgravity bioreactors [4,5], natural polymers especially collagen hydrogels [6,7,8], photopolymerized hydrogels [9], synthetic polymer scaffolds [10,11], self-assembling peptide scaffolds [12], and micro/nano patterned substrates [13]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
