Lab-on-a-chip devices, such as multielectrode arrays (MEAs), offer great advantages to study function and behavior of biological cells, such as neurons, outside the complex tissue structure. Nevertheless, in vitro systems can only succeed if they represent realistic conditions such as cell organization as similarly found in tissues. In our study, we employ a co-culture system of neuron-like (SH-SY5Y) and glial-like (U-87 MG) cells with various neuron-glial ratios to model different brain regions with different cellular compositions in vitro. We find that cell behavior in terms of cellular organization, as well as proliferation, depends on neuron-glial cell ratio, as well as the underlying substrate material. In fact, nanocolumnar titanium nitride (TiN nano), which exhibits improved electric properties for neural recording on MEA, shows improved biocompatible features compared to indium tin oxide (ITO). Moreover, electrochemical impedance spectroscopy experiments allow us to monitor cellular processes label-free in real-time over several days with multielectrode arrays. Additionally, electrochemical impedance experiments reveal superiority of TiN with nanocolumnar surface modification in comparison with ITO. TiN nano exhibits enhanced relative cell signals and improved signal-to-noise ratio, especially for smaller electrode sizes, which makes nanocolumnar TiN a promising candidate for research on neural recording and stimulation.