The Effect Of VEGF On The Responsiveness Of Voltage-gated Calcium Channels For SH-SY5Y Human Neuroblastoma Cells On Micropatterns

The three-dimensional cell culture model has been a more remarkable issuein tissue engineering and cell engineering recently. However, most of previous studiesfocused on cell growth behavior, rather than voltage-gated calcium channels (VGCCs)function and VEGF in the three-dimensional model. Therefore, this study integratedSH-SY5Y cells with PLLA patterns, and evaluated SH-SY5Y cell growth behviors andVGCCs function that were influenced by substrate morphology and VEGF.Objective This study compared differences of cell growth behavior, VGCCsfunction and growth factor secretion between PLLA planar and micropartten. Further,we evaluated difference of three-dimensional culture model compare to conventionalplanar culture models. This study laid the foundation for establishing three-dimensionalcell-based biosensing platforms.Methods UV lithography, silicon etching and soft lithography were adopted tofabricate poly-L-lactide (PLLA) planar substrates and microwell patterns. Voltage-gatedcalcium channels (VGCCs) response of SH-SY5Y cells cultured in PLLA microparttenand on flat PLLA substrates was detected by confocal microscopy with a calciumfluorescent indicator (Calcium Green-1, AM). VEGF secretion level of SH-SY5Y cellsin the micro-structure were evaluated by ELISA. And this study evaluated the effect ofVEGF on the responsiveness of VGCCs for SH-SY5Ycells.Results It was found that SH-SY5Y cells distributed on patterns’s top surfaceshowed two-dimensional (2-D) adhesion behavior, while those cells attached to themicrowell’s side-wall and bottom surface showed three-dimensional (3-D) and neartwo-dimensional (N2-D) adhesion behaviors, respectively. Microwell patterns (welldiameter:100μm, channel width:0μm and20μm) established3-D micro-environmentfor cell growth. In response to50mM high K+depolarization, responding ofpercentages or magnitudes of3-D and N2-D cells on PLLA micropartten with positiveVGCCs response was significantly lower than that for2-D cells. VEGF expression levelof SH-SY5Y were deteced by ELISA in PLLA planar structure and micropatterns, itwas found VEGF secretion level of SH-SY5Y cell in microwell patterns, significantlyhigher than those at the planar substrates. And VEGF significantly inhibited the VGCCsfunctions of SH-SY5Y cells.Conclusion PLLA microwell patterns help SH-SY5Y cell growth in three-dimensions. Through studying of VGCCs function and VEGF secretion inmicro-patterns, it was found that VGCCs function and VEGF secretion of cells withinPLLA microwells was indeed different from that of cells on planar PLLA surfaces.Furthermore, VEGF significantly inhibited the VGCCs functions of SH-SY5Y cells.Overall, these results suggested that PLLA micropattern and VEGF might be criticalfactors in affecting VGCCs responsiveness of SH-SY5Y cells. Microwell patterns couldbe an effective way for engineering3-D microenvironment for neural cell culture andestablishing3-D cell-based biosensing platforms.