Morphological Analysis of Cortical Neurons Cultured on Carbon Nanotubes and Nanofibers

Date(s) - 06/24/2013
10:00 am

Valentina Rapazzetti, BME Masters Student

In this work we investigated viability and neurite outgrowth of neuronal cells onto topographically and chemically modified surfaces. In particular purified carbon nanotubes (CNTs), electrospun SU8 carbonized (CNFs) and non-carbonized nanofibers (SU8NFs), flat stripes of SU8 thin film carbonized (CTFs) and non-carbonized (SU8TFs), and polyethylenimine (PEI) have been used, individually or in combination, as surface modifications. CNTs have been applied onto glass coverslips through a layer by layer (LBL) deposition method in which layers of PEI and CNT solution were deposited alternated on the surface. Varying the number of layers, we fabricated two types of CNT-samples: 3 layers (3LBL) and 9 layers (9LBL). All CNT samples were plasma treated after manufacturing. On the other end, the CNFs, SU8NFs, CTFs and SU8TFs were deposited on silicon substrates, plasma treated and coated with PEI at different concentrations, 0.1-0.001-0.00001 %. Dissociated E18 rat cortex neurons have been seeded on these substrates. Using calcein AM and ethidium bromide (EthD-1), cells have been stained and fluorescence microscopy applied. The images obtained were processed using NeuriteQuant, an ImageJ plugins, for cell counting and neurite extension measurements. Cell viability and neurite outgrowth was found to be higher on 9LBL samples when compared to 3LBL samples whereas no remarkable differences were detected when comparing 9LBL to control samples. For the CNFs, SU8NFs, CTFs and SU8TFs tested, very few cells grew on them at concentration of 0.1 % PEI instead high viability and neurite outgrowth was found on these samples coated with 0.001 % and 0.00001 % PEI. The results obtained can be linked with an increase in roughness, when moving from CNT to CNF and SU8NF substrates, that could have improved cell adhesion. Moreover the fact the good cell adhesion and development was detected also on the flat CTF and SU8TF samples makes us think that this material can have important biocompatible properties by itself, independently from the roughness of the surface. Another important finding of this work is that, if 0.1% PEI concentration allows cell growth and adhesion on CNTs, it prevents cell development and attachment on CNFs and SU8NFs. At the same time, a lower concentration of PEI (especially 0.00001% PEI) is very successful in enhancing neurons proliferation on both SU8NF (carbonized and uncarbonized) and on SU8TF (carbonized and uncarbonized). Nonetheless deeper analysis of samples surface chemistry and of cells activity would be needed to define an exact explanation and interpretation of the results.