Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid)

Abstract

Ze-Zhi Wu,1 Zheng-Wei Wang,1 Li-Guang Zhang,1 Zhi-Xing An,1 Dong-Huo Zhong,1 Qi-Ping Huang,1 Mei-Rong Luo,1 Yan-Jian Liao,1 Liang Jin,1 Chen-Zhong Li,2 William S Kisaalita31Key Laboratory of Biorheological Science and Technology of the State Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, People’s Republic of China; 2Nanobioengineering/Bioelectronics Laboratory, Department of Biomedical Engineering, Florida International University, Miami, Florida, 3Cellular Bioengineering Laboratory, College of Engineering, University of Georgia, Athens, Georgia, USAIntroduction: In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs).Methods and materials: SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells’ location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells’ VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium GreenTM-1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY.Results: It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K+. This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness.Conclusion: These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds.Keywords: replica molding, cell spreading, confocal microscopy, microwell patterns