Diphenylalanine Peptide Nanowires as a Substrate for Neural Cultures

Anne B Walls,Helle S Waagepetersen,Jaime Castillo-León,Małgorzata M Swiniarska,Winnie E Svendsen,Tanya Korsgaard,Maria Dimaki

doi:10.1007/s12668-019-00717-w

Abstract

Primary brain cells cultured on flat surfaces, i.e., in a two-dimensional fashion, have a long history of use as an experimental model system in neuroscience research. However, it is questionable to which extent these cultured brain cells resemble their in vivo counterparts. Mainly, it has been claimed that the non-oxidative glucose metabolism reflected by lactate production is unphysiologically high. Furthermore, it is known that culturing in 2D alters the phenotype of cells. Here we present diphenylalanine peptide nanowires (PNWs) as a culturing substrate for primary neocortical neurons from mice. The topology of the PNWs leads to neuronal cultures developing in 2.5D environment and hence improved culturing conditions. We investigate the effect of different concentrations of PNWs and different cell densities of neurons on the culturing conditions. The neocortical neurons were examined through scanning electron microscopy in order to study the effect of PNW concentrations and neuron densities on the structural appearance of the cells. Then employing the optimal combination of neuron density and PNW concentration, the neurons were evaluated functionally and metabolically by comparison with neocortical neurons standard culturing methods in 2D. Specifically, we tested neuronal viability, capacity for vesicular release of neurotransmitter GABA, as well as oxidative and non-oxidative glucose metabolism. It was evident that neurons cultured on PNWs exhibited increased viability combined with an increased capacity for neurotransmitter release and a lower fraction of non-oxidative metabolism than neurons cultured in 2D. Hence, neocortical neurons cultured in 2.5D on PNWs appear to be healthier and less glycolytic than neurons cultured in 2D.

Highlights

Primary neuronal cell cultures have long been one of the key elements in neuroscience research
At a density of 1.75 × 106 cells per ml, the neurons appeared morphologically healthier, and the cell distribution on the surfaces was more homogeneous compared to cells plated at a density of 2.5 × 106 cells per ml. Plating with this higher density resulted in the cells being washed off during the fixation procedure, and the lower cell density was chosen for the subsequent characterization of neurons cultured on peptide nanowires (PNWs) and polystyrene, respectively
We have demonstrated that neocortical neurons experience better growth condition cultured on PNWs; we found an optimal density of 1.75 × 106 cells per ml cultured on 50 mg/ml PNWs provided the most favorable culturing conditions for neocortical neurons

Summary

Introduction

Primary neuronal cell cultures have long been one of the key elements in neuroscience research. The cells are cultured using either culturing flasks or Petri dishes [1,2,3]. This is changing, and over the last decades, there has been an increased focus on developing systems that mimic the in vivo environment of cells. This is being achieved both by introducing lab-on-a-chip systems for culturing and by developing new culturing substrates. A simple and widely used method is to culture cells in a gel matrix [4,5,6]. By culturing in 2.5D on substrates with an alternative topology like for example membranes [7,8,9,10], nanostructures [11, 12], or even pillars [13,14,15,16], some of the benefits of 3D culturing can be obtained while providing the possibility of conventional post-culture

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Results

Conclusion