Evaluation of silicon nitride as a substrate for culture of PC12 cells: an interfacial model for functional studies in neurons

Silicon nitride is a biocompatible material that is currently used as an interfacial surface between cells and large-scale integration devices incorporating ion-sensitive field-effect transistor technology. Here, we investigated whether a poly-L-lysine coated silicon nitride surface is suitable for the culture of PC12 cells, which are widely used as a model for neural differentiation, and we characterized their interaction based on cell behavior when seeded on the tested material. The coated surface was first examined in terms of wettability and topography using contact angle measurements and atomic force microscopy and then, conditioned silicon nitride surface was used as the substrate for the study of PC12 cell culture properties. We found that coating silicon nitride with poly-L-lysine increased surface hydrophilicity and that exposing this coated surface to an extracellular aqueous environment gradually decreased its roughness. When PC12 cells were cultured on a coated silicon nitride surface, adhesion and spreading were facilitated, and the cells showed enhanced morphological differentiation compared to those cultured on a plastic culture dish. A bromodeoxyuridine assay demonstrated that, on the coated silicon nitride surface, higher proportions of cells left the cell cycle, remained in a quiescent state and had longer survival times. Therefore, our study of the interaction of the silicon nitride surface with PC12 cells provides important information for the production of devices that need to have optimal cell culture-supporting properties in order to be used in the study of neuronal functions

PC12 cell adhesion to an Si₃N₄ surface.

<p>PC12 cells were seeded at the same concentration on Si₃N₄ surfaces coated with different cell adhesion molecules and images of representative areas were captured using an inverted microscope 24 hours later. Non-coated Si₃N₄ surfaces were used as a control. (A) Non-coated Si₃N₄ surface, (B) concanavalin A and (C) 0.01% poly-L-lysine coated Si₃N₄ surface. (Scale bar: 50 µm).</p

Non-NGF stimulated PC12 cell proliferation on different substrates.

<p>PC12 cells were seeded on 35 mm² poly-L-lysine coated Si₃N₄ surfaces (○) or plastic culture dishes (▵) and counted every 2 days. The cells were cultured in DMEM +10% FBS +10% horse serum. The medium was replaced every 3 days. Values are the mean ± S.E. for twenty samples (Two trials of ten samples each). *, <i>p<</i>0.001 <i>vs.</i> plastic culture dish.</p

Surface structure analysis of an Si₃N₄ surface under different coating conditions.

<p>Three-dimensional atomic force microscope height images. (A) 0.01% poly-L-lysine coated plastic dish, (B) non-coated Si₃N₄ surface, (C) 0.01% poly-L-lysine coated Si₃N₄ surface at 1 day after coating and (D) at 5 days after coating, (E) 0.05% poly-L-lysine coated Si₃N₄ surface at 1 day after coating and (F) at 5 days after coating.</p

PC12 cell differentiation on a poly-L-lysine coated Si₃N₄ surface after NGF stimulation.

<p>PC12 cells expressing DsRed2 protein were seeded onto poly-L-lysine coated Si₃N₄ surfaces and 24 hours later (considered time for cell attachment), NGF (50 ng/ml) was added to the medium in the absence of FBS and horse serum. Images were captured using a fluorescence microscope at (A) 1 day, (B) 3 days, (C) 5 days and (D) 7 days after NGF stimulation. Scale bar: 100 µm.</p

Analysis of the effect of poly-L-lysine coating on contact angles.

<p>(A) Substrates were coated with poly-L-lysine solution for 2 hours and then rinsed with distilled water. After overnight drying (∼16 hours) at room temperature, images were captured using a contact angle analyzer. The angle was calculated using the instrument software and the values shown represent the averages of the angles from both sides of the droplet. Non-coated surfaces were also tested for comparative purposes. Each picture is representative for each sample group. The light square observed in the center of the droplet of the pictures taken to the plastic dish samples corresponds to the area where light passes perpendicularly through the water so that refraction does not happen, a similar effect than the one produced by a spherical lens when illuminated. It is important to mention that the light source was located at the opposite side of the water droplet in relation to the camera lens. (B) Plastic culture dishes and Si₃N₄ surfaces were left untreated or coated with 0.01% or 0.05% poly-L-lysine. A contact angle analyzer was used to measure sessile drop contact angles. Values are the mean ± S.E. for twenty samples (Two trials of ten samples each). *, <i>p<</i>0.001 <i>vs.</i> plastic culture dish.</p

Growth of extensions in PC12 cells after NGF stimulation.

<p>PC12 cells seeded on different substrates were stimulated with NGF (50 ng/ml) in the absence of FBS and horse serum and images were captured every 2 days beginning at 3 days after NGF stimulation. Using AquaCosmos analysis software, the areas of differentiated cell bodies were compared to the areas of the outgrowth extensions. Representative data are shown in the graph. Cells cultured on an Si₃N₄ surface (○ ) or on a plastic culture dish (▵). The values shown are the mean ± S.E. of 30 samples. (*, <i>p<</i>0.001 <i>vs.</i> plastic culture dish).</p

Effect of FBS and NGF on PC12 cell proliferation on a poly-L-lysine coated Si₃N₄ surface.

<p>PC12 cells were seeded onto poly-L-lysine coated Si₃N₄ surfaces in serum-supplemented growth medium and after 24 hours, cells were stimulated with NGF (50 ng/ml) in the presence/absence of FBS+horse serum. Cells were counted every 2 days. Three treatment groups were established: FBS (+) NGF (−) (○); FBS (+) NGF (+) (□); and FBS (−) NGF (+) (⋄). The values shown are the mean ± S.E. for twenty samples (Two trials of ten samples each). The presence of FBS significantly increased the cell proliferation rate (*, <i>p<</i>0.001 <i>vs.</i> FBS (−) NGF (+)); NGF significantly decreased the cell proliferation rate (*, <i>p<</i>0.001 <i>vs.</i> FBS (+) NGF (+)).</p