INTRODUCTION: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 Green™-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.

UV lithography,silicon etching and soft lithography were adopted to fabricate three-dimensional poly-Llactide(PLLA) microwell patterns.Human neuroblastoma(SH-SY5Y) cells were integrated with the PLLA patterns,and then cell growth behaviors and distribution were evaluated.Voltage-gated calcium channels(VGCCs) response of SH-SY5Y cells cultured in microwells and on flat PLLA substrates was detected by confocal microscopy with a calcium fluorescent indicator(Calcium Green-1,AM).It was found that SH-SY5Y cells distributed on patterns's top surface showed two-dimensional(2-D) adhesion behavior,while those cells attached to the microwell's side-wall and bottom surface showed three-dimensional(3-D) and boundary two-dimensional(B2-D) adhesion behaviors,respectively.In response to 50 mmol/L high K + depolarization,the percentage of 2-D cells on PLLA flat substrates with positive VGCCs response was 91.2%,while this percentage was 75% and 81%,for 3-D and B2-D cells,respectively,significantly lower than that for 2-D cells.These results suggested that cell adhesion behaviors might be critical factors in affecting VGCCs responsiveness of SH-SY5Y cells.Microwell patterns could be an effective way for engineering 3-D microenvironment for neural cell culture and establishing 3-D cell-based biosensing platforms.