An Alternative Method For The Development Of 3D Engineered Neuronal Cultures

Abstract

Event Abstract Back to Event An Alternative Method For The Development Of 3D Engineered Neuronal Cultures Nicolò Colistra1*, Mariateresa Tedesco2, Laura Pastorino1, Sergio Martinoia2 and Paolo Massobrio2 1 University of Genoa, Department of Informatics, Bioengineering, Robotics, System Engineering , Italy 2 University of Genova, Department of Informatics, Bioengineering, Robotics, System Engineering , Italy Motivation In the last years, new studies have been performed for developing in-vitro 3D neuronal systems1,2. The potential advantages of 3D engineered constructs are evident as they can be used as a more accurate investigational in-vitro platform than standard 2D network models1. 3D networks coupled to Micro-Electrode Arrays (MEAs), represent a powerful in-vitro model capable of better emulating in-vivo physiology. In this work, we propose an alternative method for the development of 3D in-vitro neuronal models with respect to a previous work, where the scaffold of 3D networks was realized by means of glass micro-beads1. Here, 3D neuronal constructs were developed and implemented by using layers of chitosan micro-beads, ranging in diameters from 40 to 80 µm, depending on the fabrication procedure. In particular, two types of chitosan were used: neutralized chitosan and cross-linked chitosan tripolyphosphate (CS/TPP). Then, the so obtained multilayered structure was coupled to planar MEAs to record the electrophysiological activity of the network. We studied the dynamics of such 3D hippocampal neuronal networks comparing their spontaneous activity with the ones of two specific control systems: (i) 3D hippocampal neuronal network grown onto glass micro-beads and (ii) standard 2D networks, both coupled to MEAs1. In addition, we analyzed the spontaneous activity of another type of 3D neuronal culture: 3D cortical neuronal network assembled onto cross-linked CS/TPP micro-beads based scaffold. Material and Methods Cell culture. Hippocampal and cortical neurons were dissociated from rats E18 Sprague Dawley. MEAs supports and all micrometric beads (made of neutralized and cross-linked chitosan) were sterilized and pre-coated with adhesion proteins (poly-lysine and laminin). Hippocampi and cortices were dissociated in a trypsin enzymatic solution and the cells were kept in a Neurobasal medium + B27 + Glutamax 1% and 1% Pen-Streptomycin. The cell suspension was deployed on preconditioned materials protein. Cultures were maintained in the incubator at 5% CO2 with a weekly change of medium. Chitosan micro-beads. Chitosan neutralized micro-beads were formed by extruding 1% w/v chitosan solution through a 0.25 mm nozzle into a basic coagulation solution consisting of sodium hydroxide-ethanol-water (20:30:50 v/v). The cross-linked micro-beads were obtained based on ionic gelation of TPP with chitosan. Briefly, in this case the chitosan was extruded into a 1% w/v TPP solution at pH6. The micro-beads were removed from the neutralizing and cross-linking solutions by centrifugation followed by four washing steps in distilled water. The so obtained microspheres were finally sterilized in 70% ethanol for 30 minutes. Data and Statistical analysis. Data analysis was performed off-line by using a custom software package named SpyCode3 developed in MATLAB (The Mathworks, Natick, MA, USA). We characterized the spontaneous activity by means of first-order statistics like mean firing rate, percentage of random spike, mean bursting rate, burst duration, network burst duration, mean network bursting rate. Data were expressed as mean ± standard error of the mean. Statistical analysis was performed using MATLAB. Since data do not follow a normal distribution, we performed a non-parametric Mann-Whitney U-test. MEA set-up. The electrophysiological activity of hippocampal (18-21DIV) and cortical neuronal networks (27-32DIV) was recorded in all the performed experiments by means of planar TiN 60 channels MEAs, supplied by Multi Channel Systems (MCS, Reutlingen, Germany). Results We analyzed the spontaneous electrophysiological activity of 3D neutralized chitosan hippocampal neuronal network and 3D cross-linked CS/TPP hippocampal neuronal network compared with the two gold-standard reference models (i.e., 2D and 3D with glass micro-beads). We evaluated the percentage of random spikes and the mean firing rate in all (n=7) the performed experiments (Figure 1). We observed that 2D networks exhibit a low percentage of random spikes (10.4 ± 3.8 %) with a high frequency of firing (12.9 ± 7.1 spikes/s), differently the 3D networks show a higher level of random spikes with a lower firing rate. In particular, the 3D neutralized chitosan network presents a significant higher level of random spikes (40.7 ± 1.5 %) but a very low frequency (2.3 ± 1 spikes/s). To quantify the bursting activity at the single channel level, the frequency and the duration of the bursts were evaluated in all the performed experiments. 2D networks present a strong synchronous bursting activity with a higher bursting rate (21.5 ± 9.8 burst/min) than the three 3D neural networks. In addition, we observed that 3D neutralized chitosan network exhibits a burst duration (311.7 ± 54.3 ms) higher than other cultures. Moreover, the spontaneous activity of 3D cross-linked CS/TPP cortical neuronal network was characterized and compared with the standard 2D cortical culture. Figure 2 shows the percentage of random spikes and the mean firing rate evaluated in n=4 experiments. We observed that 3D cortical networks show a higher level of random spikes (37 ± 11.6 %) with a lower frequency (2.3 ± 1.4 spikes/s) than the 2D ones. Conclusion The developed experimental paradigm can constitute an alternative 3D in-vitro neuronal model to elucidate neurophysiology mechanisms in in-vitro systems, and to investigate the computational properties of neuronal networks.