Establishment Of Microfluidics Platform For Compartmentalized Neuronal Culture With Axonal Propagation Velocity Measurement
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1
University of Tampere, Faculty of Medicine and Life Sciences, Finland
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2
Tampere University of Technology, Faculty of Biomedical Sciences and Engineering, Finland
Motivation
Axonal damage is one of the major hallmarks in most neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS) [1, 2]. To target the mechanisms underlying axonal damage, it is essential to study axonal physiology, pathology and electrophysiology of the nervous system in vitro because of the limitations and challenges linked to animal models. However, this can be complex due to the random orientation of axonal network and presence of cell bodies within the axonal network in neuronal cultures in vitro. To overcome these issues, microfluidics in vitro models for axonal damage have been suggested [3-7]. Microfluidics technology enable generation of multi-compartment cell culture platforms that allow compartmentalized cell culture and guidance of cell growth [7-8]. This leads to possibility for studying axons in isolation and guiding axons to align on the microelectrode array (MEA) electrodes resulting in amplified neuronal activity [9-11]. Reported microfluidics platforms for axonal electrophysiology have been established for rodent neurons [3-6]. As the behavior of rodent and human neurons can differ, studies should be directed also on human cells. The aim of this study was to establish a microfluidic platform for human-derived neurons with isolated axonal culture that can be used for measuring the propagation velocity of action potential.
Material and Methods
A microfluidics platform was fabricated from polydimethylsiloxane (PDMS) using soft lithography techniques [12]. The platform contains separate compartments for neuronal somas and axons and compartments are interconnected by microtunnels. To achieve compartmentalization of somas and axons, variable dimensions for microtunnels were tested; a height of 1 µm and 3.5 µm, a length of 100 µm, 250 µm and 500 µm, and a width of 10 µm. Human pluripotent stem cell (hPSC) –derived cortical neurons were cultured in the platform. hPSCs were cultured in feeder-free conditions using Essential 8™ medium (Thermo Fisher Scientific, USA) and laminin-521 coating (Biolamina, Sweden) [13]. The neural differentiation of hPSCs was conducted in adherent culture using dual smad inhibition method modified from earlier protocols [14]. The growth, migration and behavior of neurons in the microfluidics platform was followed using a phase contrast microscope. To evaluate the number of neuronal somas that enter through the microtunnels into the axon compartments, nuclear staining using 4',6-diamidino-2 phenylindole (DAPI) for living cells was used. To determine the viability of the cells and to verify the neuronal identity of the cells in the microfluidics platform, a LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells (Thermo Fisher Scientific) and immunocytochemical staining with antibodies against MAP-2, β-tubulin and GFAP was performed. In order to study the propagation velocity of action potential of hPSC-derived neurons in the microfluidics platform, the platform was integrated on the commercial standard MEA (Multi Channel Systems, Germany). Spontaneous activity of cells was followed with MEA recording twice a week for up to 35 days and the velocity was estimated manually from the signals measured in the microtunnels. At the end point (day 35), pharmacological treatments using glutamate and tetrodotoxin (TTX) were performed for the cultures in the platform and responses were measured by MEA recording.
Results
Axonal growth through the microtunnels of different sizes was followed by monitoring the status of axons by phase-contrast imaging every second or third day up to 16 days. There was a clear trend that the penetration of axons though the microtunnels was height- and length-dependent. Phase-contrast imaging also revealed that even though axons traversed the microtunnels of 1 µm height, their number in the axon compartment was minor compared to axons that traversed the microtunnels of 3.5 µm height. Furthermore, axons passing through the 1 µm high microtunnels were unable to extend in the axon compartment, whereas axons passing through the 3.5 µm high microtunnels extended normally. DAPI staining showed that neuronal somas were unable to penetrate through the 1 µm high microtunnels but the penetration of somas through the 3.5 µm high microtunnels was length-dependent. LIVE/DEAD assay showed that neuron culture in the platform was viable and immunocytochemical staining the neuronal nature of the cells. The propagation velocity of action potential of hPSC-derived neurons in the platform was measured in the range of 0.8-2 m/s during the five weeks culturing period. Glutamate treatment caused a temporal, network-wide increase in neuronal activity within the platform and TTX treatment inhibited neuronal signaling gradually.
Discussion
Microfluidics-based models that have so far been established for axonal electrophysiology have utilized rodent neurons [3-6]. As results from rodent cells cannot be translated directly into proven fact of human cells, mechanisms related to human diseases are important to study using human cells. In this study, we optimized and established a microfluidics platform that can be used for measuring the propagation velocity of action potential of hPSC-derived neurons. In the platform, axons were isolated from their cell somas with optimized microtunnel size. To amplify axonal activity and ease its detection [9-11], the growth of axons was restricted to the vicinity of MEA electrodes with the help of microtunnels and compartmentalization of the platform. The platform was proven to be functional as the propagation velocity was measured within the biological range [15] and neurons were healthy, viable and verified as neuronal population. The established platform was designed for commercial MEA, thus enabling its wider use in the field of axonal electrophysiology.
Conclusions
The established microfluidics platform is suitable for the culture of hPSC-derived neurons enabling axonal isolation and action potential measurement. The platform has potential use in the field of axonal biology and axonal electrophysiology.
Acknowledgements
The authors acknowledge The Academy of Finland (grant number 296415 for MR), Finnish Funding Agency for Technology and Innovation, Juliana von Wendt Foundation and the Finnish MS Foundation for financial support and Dr. Tina Stummann (Lundbeck A/S, Denmark) for contribution in establishing neural differentiation methods.
References
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Keywords:
Microfluidics,
Neuron,
Human pluripotent stem cell,
axonal network,
action potential velocity
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Poster Presentation
Topic:
Microphysiological systems
Citation:
Ristola
M,
Sukki
L,
Tanner
A,
Hyvärinen
T,
Rad
AB,
Ylä-Outinen
L,
Kallio
P and
Narkilahti
S
(2019). Establishment Of Microfluidics Platform For Compartmentalized Neuronal Culture With Axonal Propagation Velocity Measurement.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00051
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Received:
22 May 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Dr. Mervi Ristola, University of Tampere, Faculty of Medicine and Life Sciences, Tampere, Finland, mervi.ristola@uta.fi