Design in Neuronal Dissociated Cultures for Functional Integration of Cell Populations
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1
Lobachevsky State University of Nizhny Novgorod, Department of Neuroengineering, Center of Translational Technologies, Russia
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2
Saint-Petersburg Academic University, Nanotechnology Research and Education Centre of the RAS, Russia
Motivation
Microfluidics combined with microelectrode array technology recently have been used in neuroengeneering to study fundamental mechanisms of the brain and in medical applications. Dissociated neuronal cultures grown on microelectrode arrays allows continuous long term registration and electrical stimulation of the neuronal network. Microfluidic technology allows to grow neural branches (axons and dendrites) inside microchannels which separates cellular clusters. We combined these methods to study interaction between progenitor cells and mature neural tissue [1]. We designed specific microchannels structure that provided functional and morphological integration of primary hippocampal culture with cells from E14 neurospheres.
Material and Methods
Microfluidic device
PDMS microfluidic chips containing an array of microchannels between two chambers A and B were fabricated by means of two-layer lithography and PDMS molding techniques. Channel design determined axonal growth dominantly from A chamber to B. Mold design contained: first 5 mkm thick layer, which formed microchannel structure and second 50 mkm-thick layer, which formed chambers. Microchannels’ structure consisted of three 200 mkm segments with 7 mkm bottlenecks, total length was 600 mkm. In order to investigate spike propagation inside the microchannels, each PDMS chip was positioned and mounted onto the surface of a planar microelectrode array (MEA), so as to locate several electrodes in the microchannels. Before cell plating the device was pre-treated with the adhesion promoting molecule polyethyleneimine (Sigma P3143).
Culture preparation
Hippocampal cells were dissociated from embryonic mice (E18) and plated into separate left chamber on MEAs at a final density of approximately 15,000–20,000 cells/mm2. After 10 days in in vitro E14 forebrain cells were plated into right chamber. E14 forebrain cells were preliminary cultivated in serum free medium (DMEM F12 with FGF – 50 ng/ml, EGF – 50 ng/ml, N2 – 10 mkl/ml, 1% B27 and 1% L-glu) during 4 days forming neurospheres. The neurospheres were dissociated with 0,25% trypsin (5min) and mechanical trituration and plated in the right chamber of PDMS chip (fig. 1,2) with a final density of approximately 20,000 cells/mm2.
Results
In this study we designed a microfluidic device combined with microelectrode array which allows growing two separate neuronal cells cultured networks with directed functional pathways between. We tested the device for functional integration of two different cell populations. Spontaneous and stimulus evoked bursts in the network of E18 hippocampus cells propagated in the right chamber and initiated bursting activity in the network of E14 cells. Efficacy of the burst propagation was about 90% (fig/ 3). It shows good functional integration of two networks.
Conclusion
Developed devise allowed functionally integrate primary hippocampal culture and cells from E14 neurospheres with good efficacy. Designed microchannels structure can be used to investigate progenitor cell differentiation and functional integration in the network. The results also can be used in the study of neuroprostethics, neuroreabilitation and synaptic plasticity.
References
[1] Scadden D.T. The Stem-Cell Niche as an Entity of Action. Nature 2006.441 (7097): 1075–79
[2] Pimashkin A. Kastalskiy I, Simonov A, Koryagina E, Mukhina I, Kazantsev V. Spiking signatures of spontaneous activity bursts in hippocampal cultures. Front. Comput. Neurosci 2011; 5(November): 46.
Fig. 1. Experimental protocol. E18 hippocampal cells were grown for 10 days. Then E14 Neurospheres from hippocampal progenitor cells was plated in right chamber and then grown for 2-3 weeks.
(see Methods).
Fig. 2. Neuronal cultures grown on microelectrode array. (A) Left chamber – hippocampal neurons (E18) 17DIV, axons growed to left chamber through microchannels. (B) Left chamber – E18 celll 28DIV, left chamber – E14 progenitor neurons diffirentiated and growed for 10 days. Bioelectrical bursting activity from E14 progenitor neurons appeared on 10DIV.
Fig. 3. Time histogram of average network spontaneous burst profile. In average, spikes from Left chamber (E18, blue line) propagated through axons in microchannels (red line) and evoked bursts in E14 neurons in the Right chamber (green).
Acknowledgements
This research was supported by the Russian Science Foundation (¹ 14-19-01381).
Keywords:
Microfluidics,
Neurons,
development,
neurospheres,
Dissociated cultures,
Network bursting
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
Poster Presentation
Topic:
MEA Meeting 2016
Citation:
Gladkov
A,
Pigareva
Y,
Kolpakov
V,
Malishev
E,
Bukatin
A,
Kazantsev
V,
Mukhina
I and
Pimashkin
A
(2016). Design in Neuronal Dissociated Cultures for Functional Integration of Cell Populations.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00084
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Arseniy Gladkov, Lobachevsky State University of Nizhny Novgorod, Department of Neuroengineering, Center of Translational Technologies, Nizhny Novgorod, Russia, gladkov@neuro.nnov.ru