| The interaction between cells and substrates is a common concern in cell biology,biomaterials and tissue engineering.It is important to explore and imitate the growth and functional responses for neural cells in specific microenvironment by microfabrication technology with the purpose of understanding the biological behavior of embryos development,tissue regeneration and repair in vivo.However,some patterns had a low aspect ratio or depth,which the cells growing on the flat bottom surface restricted by sidewalls did not form any 3D distribution or along the depth of sidewalls.Actually,these patterns could only be defined as near two-dimensional(N2D)culture systems with limited boundaries.Thus,it is necessary to determine whether such N2 D culture systems are different from 3D culture systems with high depth sidewalls in functional behaviors of cells growing therein at single cell level.In addition,little is known about the responses at cell-substrate or cell-cell interface to topographical cues with 3D multiple cells,though there have been lots of cellular aggregates cultured within micro-wells mainly for neural stem cells(NSCs)culture in vitro.Therefore,it is essential to construct multi-dimensional culture of neural cells at different cell levels on patterned structures and evaluate the relationship between functional and growth behavior of cells in situ for neural cell-based microsystem.Thus,the patterned substrates consist of arrayed cylindrical micro-wells with or without channels connected were designed and fabricated with biodegradable polymer PLLA based on the microfabrication technology in this study.These patterns had a nominal diameter of 80 ?m,100 ?m,120 ?m micro-well,either with or without channels of 20 or 40 ?m in width,and the depths were uniform 100 ?m.And then the SH-SY5 Y human neuroblastoma cells and hippocampal neural stem cells(NSCs)were interfaced with the patterns and induced differentiation,respectively.And the two multi-dimensional culture systems model for neural cells with different cellular levels were constructed by the cells directed migration on the patterns,and the influential factors were also investigated.In addition,the growth characteristics and functional responsiveness of cells were evaluated on these patterns based on immunofluorescence staining and calcium ion detection technology,and the regulation mechanism of ROCK signaling pathway and RGK protein Gem in the growth behaviors and functional responses of cells was explored.This study will be applied to construct the neural cell-based microsystems in high-throughput drug screening and target response evaluation and tissue engineering implant with functional neural networks in vitro.The main contents and results of present study are as follows:(1)The poly-L-lactic acid(PLLA)micro-well patterned substrates were designed and fabricated to construct the SH-SY5 Y cells multi-dimensional culture systems model and evaluate the growth characteristics and functional responses of calcium channels at single cell level.The growth characteristics of SH-SY5 Y cells on the patterns could be classified as near two-dimensional(N2D),three-dimensional(3D)and two-dimensional(2D)culture systems according to their location on the patterns.And the construction of multi-dimensional culture systems model was accomplished by cells directed migration induced by the asymmetric structural features,accompanied by the rearrangement of cell cytoskeleton and morphological characteristics of cells.Cells within micro-wells(N2D and 3D cells)showed a decreased projection areas and VGCCs responsiveness compared to FS-2D cells on flat substrates,and N2 D cells consistently had an intermediate value between those for FS-2D and 3D cells,suggesting a correlative relation between cell morphology and VGCCs responsiveness.(2)Then,the mechanism of growth and functional responses of SH-SY5 Y cells on the patterned substrates was investigated by ROCK signal pathway inhibitor.It was found that the rearrangement of microfilament cytoskeleton,morphological spreading and assembly of focal adhesions for cells on the patterns was suppressed through the ROCK signal pathway down-regulated by the patterns.However,the VGCCs responsiveness of FS-2D or N2 D cells with spread morphology were suppressed by the inhibition of ROCK signal pathway,while promoted for 3D cells on the sidewalls with spherical morphology,and this result was associated with the expression of Gem.The results indicated that the functional response of cells is closely related to its culture system and morphological characteristics.(3)The hippocampal neural stem cells(NSCs)were cultured on the micro-well patterns to construct the multi-dimensional culture systems model and evaluate its influential factors at multiple cells level.It was found that the construction of NSCs multi-dimensional culture model was regulated by cells directional migration through the interactions of cell-substrate or cell-cell interface for NSCs on patterned substrates,which cells within micro-wells were mainly cultured with three-dimensional cellular aggregates(MW-3D cells)and cells on the top surfaces were mainly cultured with two-dimensional single-layer adherent culture(TS-2D cells).And the patterns without channels connection were earlier than patterns with channels connection for the construction of this model.Indeed,the proliferation and stemness maintenance of NSCs within micro-wells was promoted by the patterns with channels connection as the suitable microenvironment for a long time,and it can be used to build quasi-one-dimensional neural network within micro-wells.(4)Then,the differentiation of hippocampal NSCs were induced on the patterns to construct the patterned neural networks and evaluate the functional differentiation.It was found that the structural features of patterned substrate played an important role in the migration of cells,extension of neurite outgrowth and orientation of nucleus,and the MW-3D cells could be regarded as "seed source" in the process of patterned neural network construction by cells migration along the sidewalls.And it realized the functional differentiation of cells in the constructed neural networks,such as MAP-2 positive dendrites and VGCCs functional characteristics of typical neurons. |
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